Microporation of tissue for delivery of bioactive agents

ABSTRACT

A method of enhancing the permeability of a biological membrane, including the skin or mucosa of an animal or the outer layer of a plant to a permeant is described utilizing microporation of selected depth and optionally one or more of sonic, electromagnetic, mechanical and thermal energy and a chemical enhancer. Microporation is accomplished to form a micropore of selected depth in the biological membrane and the porated site is contacted with the permeant. Additional permeation enhancement measures may be applied to the site to enhance both the flux rate of the permeant into the organism through the micropores as well as into targeted tissues within the organism.

BACKGROUND OF THE INVENTION

[0001] This invention relates generally to the field of transmembranedelivery of drugs or bioactive molecules to an organism. Moreparticularly, this invention relates to a minimally invasive tonon-invasive method of increasing the permeability of the skin, mucosalmembrane or outer layer of a plant through microporation of thisbiological membrane, which can be combined with sonic, electromagnetic,and thermal energy, chemical permeation enhancers, pressure, and thelike for selectively enhancing flux rate of bioactive molecules into theorganism and, once in the organism, into selected regions of the tissuestherein.

[0002] The stratum corneum is chiefly responsible for the well knownbarrier properties of skin. Thus, it is this layer that presents thegreatest barrier to transdermal flux of drugs or other molecules intothe body and of analytes out of the body. The stratum corneum, the outerhorny layer of the skin, is a complex structure of compact keratinizedcell remnants separated by lipid domains. Compared to the oral orgastric mucosa, the stratum corneum is much less permeable to moleculeseither external or internal to the body. The stratum corneum is formedfrom keratinocytes, which comprise the majority of epidermal cells, thatlose their nuclei and become corneocytes. These dead cells comprise thestratum corneum, which has a thickness of only about 10-30 μm and, asnoted above, is a very resistant waterproof membrane that protects thebody from invasion by exterior substances and the outward migration offluids and dissolved molecules. The stratum corneum is continuouslyrenewed by shedding of corneum cells during desquamination and theformation of new corneum cells by the keratinization process.

[0003] Underlying the stratum corneum is the viable cell layer of theepidermis and the dermis, or connective tissue layer. These layerstogether make up the skin. Microporation of these underlying layers (theviable cell layer and dermis) has not previously been used but mayenhance transdermal flux. Deep to the dermis are the underlyingstructures of the body, including fat, muscle, bone, etc.

[0004] Microporation of the mucous membrane has not been usedpreviously. The mucous membrane generally lacks a stratum corneum. Themost superficial layer is the epithelial layer which consists ofnumerous layers of viable cells. Deep to the epithelial layer is thelamina propria, or connective tissue layer.

[0005] Microporation of plants has been previously limited to selectapplications in individual cells in laboratory settings. Plant organismsgenerally have tough outer layers to provide resistance to the elementsand disease. Microporation of this tough outer layer of plants enablesthe delivery of substances useful for introduction into the plant suchas for conferring the desired trait to the plant or for production of adesired substance. For example, a plant may be treated such that eachcell of the plant expresses a particular and useful peptide such as ahormone or human insulin.

[0006] The flux of a drug or analyte across the biological membrane canbe increased by changing either the resistance (the diffusioncoefficient) or the driving force (the gradient for diffusion). Flux maybe enhanced by the use of so-called penetration or chemical enhancers.Chemical enhancers are well known in the art and a more detaileddescription will follow.

[0007] Another method of increasing the permeability of skin to drugs isiontophoresis. Iontophoresis involves the application of an externalelectric field and topical delivery of an ionized form of drug or anun-ionized drug carried with the water flux associated with iontransport (electro-osmosis). While permeation enhancement withiontophoresis has been effective, control of drug delivery andirreversible skin damage are problems associated with the technique.

[0008] Sonic energy has also been used to enhance permeability of theskin and synthetic membranes to drugs and other molecules. Ultrasoundhas been defined as mechanical pressure waves with frequencies above 20kHz, H. Lutz et al., Manual of Ultrasound 3-12 (1984). Sonic energy isgenerated by vibrating a piezoelectric crystal or otherelectromechanical element by passing an alternating current through thematerial, R. Brucks et al., 6 Pharm. Res. 697 (1989). The use of sonicenergy to increase the permeability of the skin to drug molecules hasbeen termed sonophoresis or phonophoresis.

[0009] Although it has been acknowledged that enhancing permeability ofthe skin should theoretically make it possible to transport moleculesfrom inside the body through the skin to outside the body for collectionor monitoring, practicable methods have not been disclosed. U.S. Pat.No. 5,139,023 to Stanley et al. discloses an apparatus and method fornoninvasive blood glucose monitoring. In this invention, chemicalpermeation enhancers are used to increase the permeability of mucosaltissue or skin to glucose. Glucose then passively diffuses through themucosal tissue or skin and is captured in a receiving medium. The amountof glucose in the receiving medium is measured and correlated todetermine the blood glucose level. However, as taught in Stanley et al.,this method is much more efficient when used on mucosal tissue, such asbuccal tissue, which results in detectable amounts of glucose beingcollected in the receiving medium after a lag time of about 10-20minutes. However, the method taught by Stanley et al. results in anextremely long lag time, ranging from 2 to 24 hours depending on thechemical enhancer composition used, before detectable amounts of glucosecan be detected diffusing through human skin (heat-separated epidermis)in vitro. These long lag times may be attributed to the length of timerequired for the chemical permeation enhancers to passively diffusethrough the skin and to enhance the permeability of the barrier stratumcorneum, as well as the length of time required for the glucose topassively diffuse out through the skin. Thus, Stanley et al. clearlydoes not teach a method for transporting blood glucose or other analytesnon-invasively through the skin in a manner that allows for rapidmonitoring, as is required for blood glucose monitoring of diabeticpatients and for many other body analytes such as blood electrolytes.

[0010] While the use of sonic energy for drug delivery is known, resultshave been largely disappointing in that enhancement of permeability hasbeen relatively low. There is no consensus on the efficacy of sonicenergy for increasing drug flux across the skin. While some studiesreport the success of sonophoresis, J. Davick et al., 68 Phys. Ther.1672 (1988); J. Griffin et al., 47 Phys. Ther. 594 (1967); J. Griffin &J. Touchstone, 42 Am. J. Phys. Med. 77 (1963); J. Griffin et al., 44 Am.J. Phys. Med. 20 (1965); D. Levy et al., 83 J. Clin. Invest. 2074); D.Bommannan et al., 9 Pharm. Res. 559 (1992), others have obtainednegative results, H. Benson et al., 69 Phys. Ther. 113 (1988); J.McElnay et al., 20 Br. J. Clin. Pharmacol. 4221 (1985); H. Pratzel etal., 13 J. Rheumatol. 1122 (1986). Systems in which rodent skin wereemployed showed the most promising results, whereas systems in whichhuman skin was employed have generally shown disappointing results. Itis well known to those skilled in the art that rodent skin is much morepermeable than human skin, and consequently the above results do notteach one skilled in the art how to effectively. utilize sonophoresis asapplied to transdermal delivery and/or monitoring through human skin.

[0011] A significant improvement in the use of ultrasonic energy in themonitoring of analytes and also in the delivery of drugs to the body isdisclosed and claimed in copending application Ser. No. 08/152,442 filedNov. 15, 1993, now U.S. Pat. No. 5,458,140, and Ser. No. 08/152,174filed Dec. 8, 1993, now U.S. Pat. No. 5,445,611, both of which areincorporated herein by reference. In these inventions, the transdermalsampling of an analyte or the transdermal delivery of drugs, isaccomplished through the use of sonic energy that is modulated inintensity, phase, or frequency or a combination of these parameterscoupled with the use of chemical permeation enhancers. Also disclosed isthe use of sonic energy, optionally with modulations of frequency,intensity, and/or phase, to controllably push and/or pump moleculesthrough the stratum corneum via perforations introduced by needlepuncture, hydraulic jet, laser, electroporation, or other methods.

[0012] The formation of micropores (i.e. microporation) in the stratumcorneum to enhance the delivery of drugs has been the subject of variousstudies and has resulted in the issuance of patents for such techniques.

[0013] Jacques et al., 88 J. Invest. Dermatol. 88-93 (1987), teaches amethod of administering a drug by ablating the stratum corneum of aregion of the skin using pulsed laser light of wavelength, pulse length,pulse energy, pulse number, and pulse repetition rate sufficient toablate the stratum corneum without significantly damaging the underlyingepidermis and then applying the drug to the region of ablation. Thiswork resulted in the issuance of U.S. Pat. No. 4,775,361 to Jacques etal. The ablation of skin through the use of ultraviolet-laserirradiation was earlier reported by Lane et al., 121 Arch. Dermatol.609-617 (1985). Jacques et al. is restricted to use of few wavelengthsof light and expensive lasers.

[0014] Tankovich, U.S. Pat. No. 5,165,418 (hereinafter, “Tankovich'418”), discloses a method of obtaining a blood sample by irradiatinghuman or animal skin with one or more laser pulses of sufficient energyto cause the vaporization of skin tissue so as to produce a hole in theskin extending through the epidermis and to sever at least one bloodvessel, causing a quantity of blood to be expelled through the hole suchthat it can be collected. Tankovich '418 thus is inadequate fornoninvasive or minimally invasive permeabilization of the stratumcorneum such that a drug can be delivered to the body or an analyte fromthe body can be analyzed.

[0015] Tankovich et al., U.S. Pat. No. 5,423,803 (hereinafter,“Tankovich '803”) discloses a method of laser removal of superficialepidermal skin cells in human skin for cosmetic applications. The methodcomprises applying a light-absorbing “contaminant” to the outer layersof the epidermis and forcing some of this contaminant into or throughthe intercellular spaces in the stratum corneum, and illuminating theinfiltrated skin with pulses of laser light of sufficient intensity thatthe amount of energy absorbed by the contaminant will cause thecontaminant to explode with sufficient energy to tear off some of theepidermal skin cells. Tankovich '803 further teaches that there shouldbe high absorption of energy by the contaminant at the wavelength of thelaser beam, that the laser beam must be a pulsed beam of less than 1 μsduration, that the contaminant must be forced into or through the upperlayers of the epidermis, and that the contaminant must explode withsufficient energy to tear off epidermal cells upon absorption of thelaser energy. This invention also fails to disclose or suggest a methodof drug delivery or analyte collection.

[0016] Raven et al., WO 92/00106, describes a method of selectivelyremoving unhealthy tissue from a body by administering to a selectedtissue a compound that is highly absorbent of infrared radiation ofwavelength 750-860 nm and irradiating the region with correspondinginfrared radiation at a power sufficient to cause thermal vaporizationof the tissue to which the compound was administered but insufficient tocause vaporization of tissue to which the compound had not beenadministered. The absorbent compound should be soluble in water orserum, such as indocyanine green, chlorophyll, porphyrins,heme-containing compounds, or compounds containing a polyene structure,and power levels are in the range of 50-1000 W/cm2 or even higher.

[0017] Konig et al., DD 259351, teaches a process for thermal treatmentof tumor tissue that comprises depositing a medium in the tumor tissuethat absorbs radiation in the red and/or near red infrared spectralregion, and irradiating the infiltrated tissue with an appropriatewavelength of laser light. Absorbing media can include methylene blue,reduced porphyrin or its aggregates, and phthalocyanine blue. Methyleneblue, which strongly absorbs at 600-700 nm, and a krypton laser emittingat 647 and 676 nm are exemplified. The power level should be at least200 mW/cm².

[0018] It has been shown that by stripping the stratum corneum from asmall area of the skin with repeated application and removal ofcellophane tape to the same location one can easily collect arbitraryquantities of interstitial fluid, which can then be assayed for a numberof analytes of interest. Similarly, the ‘tape-stripped’ skin has alsobeen shown to be permeable to the transdermal delivery of compounds intothe body. Unfortunately, ‘tape-stripping’ leaves a open sore which takesweeks to heal, and for this, as well as other reasons, is not consideredas an acceptable practice for enhancing transcutaneous transport in wideapplications.

[0019] As discussed above, it has been shown that pulsed lasers, such asthe excimer laser operating at 193 nm, the erbium laser operating near2.9 μm or the CO₂ laser operating at 10.2 μm, can be used to effectivelyablate small holes in the human stratum corneum. These laser ablationtechniques offer the potential for a selective and potentiallynon-traumatic method for opening a delivery and/or sampling hole throughthe stratum corneum. However, due to the prohibitively high costsassociated with these light sources, there have been no commercialproducts developed based on this concept. The presently disclosedinvention, by defining a method for directly conducting thermal energyinto or through the biological membrane with very tightly definedspatial and temporal resolution, makes it possible to produce thedesired micro-ablation of the biological membrane very low cost energysources.

[0020] In view of the foregoing problems and/or deficiencies, thedevelopment of a method for safely enhancing the permeability of thebiological membrane for minimally invasive or noninvasive monitoring ofbody analytes in a more rapid time frame would be a significantadvancement in the art. It would be another significant advancement inthe art to provide a method of minimally invasively or non-invasivelyenhancing the transmembrane flux rate of a drug into a selected area ofan organism.

[0021] Significant advancements in the delivery of drugs and othercompounds are being made through the use of various techniques thatincrease the permeability of a biological membrane, such as the skin ormucosal membrane. Even more promising advances have been made throughtechniques for creating micropores, as disclosed in the aforementionedapplications.

[0022] Nevertheless, it is desirable to improve upon these technologiesby forming micropores at selected depths in the biological membrane andto deliver both small and large compounds, in terms of molecular weightand size, through the micropores into the body.

BRIEF SUMMARY OF THE INVENTION

[0023] This invention provides a method for enhancing the transmembraneflux rate of a permeant into a selected site of an organism comprisingthe steps of enhancing the permeability of said selected site of theorganism to said permeant by means of (a) porating a biological membraneat said selected site by means that form a micropore in said biologicalmembrane, thereby reducing the barrier properties of said biologicalmembrane to the flux of said permeant and (b) contacting the poratedselected site with a composition comprising an effective amount of saidpermeant, whereby the transmembrane flux rate of said permeant into theorganism is enhanced.

[0024] This invention further provides the method of enhancing thetransmembrane flux rate further comprising applying to said site of saidorganism an enhancer to increase the flux of said permeant into saidorganism. The invention also provides the method wherein said enhancercomprises sonic energy, and more specifically, wherein the said sonicenergy is applied to said site at a frequency in the range of about 10Hz to 1000 MHz, and wherein said sonic energy is modulated by means of amember selected from the group consisting of frequency modulation,amplitude modulation, phase modulation, and combinations thereof.Alternatively, the said enhancer comprises an electromagnetic field,and, more specifically, iontophoresis or a magnetic field., or amechanical force, chemical enhancer, or thermal enhancer. Additionally,the invention further provides a method wherein any of the methods ofsonic, electromagnetic, mechanical, thermal, or chemical enhancement maybe applied in any combination thereof to increase the transmembrane fluxrate of said permeant into or through said micropore.

[0025] This invention also provides a method of further enhancing thetransmembrane flux rate with an enhancer, wherein said enhancers at saidsite are applied so as to increase the flux rate of the permeant intotissues surrounding the micropore. The said enhancer can comprise sonicenergy. Furthermore, the said sonic energy is applied to said site at afrequency in the range of about 10 Hz to 1000 MHz, wherein said sonicenergy is modulated by means of a member selected from the groupconsisting of frequency modulation, amplitude modulation, phasemodulation, and combinations thereof. Alternatively, the said enhancercomprises sonic or thermal energy, electroporation, iontophoresis,chemical enhancers, mechanical force, or a magnetic field, or anycombination thereof.

[0026] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant further comprising applying tosaid site of said organism an enhancer, wherein any of the methods ofmethods of sonic or thermal energy, electroporation, iontophoresis,chemical enhancers, mechanical force, or a magnetic field may be appliedin any combination thereof further comprising the method of combiningsonic or thermal energy, electroporation, iontophoresis, chemicalenhancers, mechanical force, or a magnetic field to increase the fluxrate of the permeant into tissues surrounding the micropore.

[0027] The invention also includes the method of further enhancing thetranmembrane flux rate within and beneath the outer layer wherein saidporating of said biological membrane in said site is accomplished bymeans selected from the group consisting of (a) ablating the biologicalmembrane by contacting said site, up to about 1000 μm across, of saidbiological membrane with a heat source such that a micropore is formedin said biological membrane at said site; (b) puncturing said biologicalmembrane with a micro-lancet calibrated to form a micropore of up toabout 1000 μm in diameter; (c) ablating the biological membrane by abeam of sonic energy onto said biological membrane up to about 1000 μmin diameter; (d) hydraulically puncturing said biological membrane witha high pressure jet of fluid to form a micropore of up to about 1000 μmin diameter and (e) puncturing said biological membrane with shortpulses of electricity to form a micropore of up to about 1000 μm indiameter. Further, the invention includes the method wherein saidporating is accomplished by contacting said site, up to about 1000 μmacross, with a heat source to conductively transfer an effective amountof thermal energy to said site such that the temperature of some of thewater and other vaporizable substances in said site is elevated abovetheir vaporization point creating a micropore to a selected depth in thebiological membrane at said site or wherein said porating isaccomplished by contacting said site, up to about 1000 μm across, with aheat source to conductively transfer an effective amount of thermalenergy to said site such that the temperature of some of the tissue atsaid site is elevated to the point where thermal decomposition occurscreating a micropore to a selected depth in the biological membrane atsaid site. Additionally, the invention includes the method of poratingsaid biological membrane in said site further comprising treating atleast said site with an effective amount of a substance that exhibitssufficient absorption over the emission range of a pulsed light sourceand focusing the output of a series of pulses from said pulsed lightsource onto said substance such that said substance is heatedsufficiently to conductively transfer an effective amount of thermalenergy to said biological membrane to elevate the temperature to therebycreate a micropore. The invention also includes the method wherein saidpulsed light source emits at a wavelength that is not significantlyabsorbed by said biological membrane. The invention further provides themethod wherein said pulsed light source is a laser diode emitting in therange of about 630 to 1550 nm, wherein said pulsed light source is alaser diode pumped optical parametric oscillator emitting in the rangeof about 700 and 3000 nm, wherein said pulsed light source is a memberselected from the group consisting of arc lamps, incandescent lamps, andlight emitting diodes. The invention also includes the method furthercomprising providing a sensing system for determining when the microporein the biological membrane has reached the desired dimensions, includingwidth, length, and depth, and, further, wherein said sensing systemcomprises light collection means for receiving light reflected from saidsite and focusing said reflected light on a detector for receiving saidlight and sending a signal to a controller wherein said signal indicatesa quality of said light, and a controller coupled to said detector andto said light source for receiving said signal and for shutting off saidlight source when a preselected signal is received, or, alternatively,an electrical impedance measuring system which can detect the changes inthe impedance of the biological membrane at different depths into theorganism as the micropore is formed.

[0028] The invention also provides the method of enhancing thetranmembrane flux rate within and beneath the outer layer furthercomprising cooling said site and adjacent tissues such that said siteand adjacent tissues are in a cooled condition. The said cooling meanscomprises a Peltier device.

[0029] The invention also includes the method of enhancing thetransmembrane flux within and beneath the outer layer furthercomprising, prior to porating said site, illuminating at least said sitewith light such that said site is sterilized.

[0030] This invention also includes the method of enhancing thetransmembrane flux within and beneath the outer layer further comprisingcontacting said site with a solid element, wherein said solid elementfunctions as a heat source to conductively transfer an effective amountof thermal energy to said biological membrane to elevate the temperatureto thereby create a micropore. Further, said heat source is constructedto modulate the temperature of said site to greater than 100° C. withinabout 10 nanoseconds to 50 milliseconds and then returning thetemperature of said site to approximately ambient temperature withinabout 1 millisecond to 50 milliseconds and wherein a cycle of raisingthe temperature and returning to ambient temperature is repeated one ormore times effective for porating the biological membrane to the desireddepth. The invention further includes the method of using a heat sourcewherein said returning to approximately ambient temperature of said siteis carried out by withdrawing said heat source from contact with saidsite and wherein the modulation parameters are selected to reducesensation to the animal subject.

[0031] The invention includes the method for enhancing transmembraneflux rates using a heat source and sensing system further comprisingproviding means for monitoring electrical impedance between said solidelement and said organism through said site and adjacent tissues andmeans for advancing the position of said solid element such that as saidporation occurs with a concomitant change in impedance, said advancingmeans advances the solid element such that the solid element is incontact with said site during heating of the solid element, until theselected impedance is obtained. Further, the invention includes thismethod further comprising means for withdrawing said solid element fromcontact with said site wherein said monitoring means is capable ofdetecting a change in impedance associated with contacting a selectedlayer underlying the surface of said site and sending a signal to saidwithdrawing means to withdrawn said solid element from contact with saidsite.

[0032] The method of enhancing the transmembrane flux rate using a solidelement wherein said solid element is heated by delivering an electricalcurrent through an ohmic heating element and, further, wherein saidsolid element is formed such that it contains an electrically conductivecomponent and the temperature of said solid element is modulated bypassing a modulated electrical current through said conductive element.Additionally, the invention includes the method wherein said solidelement is positioned in a modulatable magnetic field wherein energizingthe magnetic field produces electrical eddy currents sufficient to heatthe solid element.

[0033] The invention also includes the method of enhancing thetransmembrane flux rate wherein said porating is accomplished bypuncturing said site with a micro-lancet calibrated to form a microporeof up to about 1000 μm in diameter, by a beam of sonic energy directedonto said site to form a micropore of up to about 1000 μm in diameter,by hydraulically puncturing said biological membrane with a highpressure jet of fluid to form a micropore of up to about 1000 μm indiameter, or, alternatively, by puncturing said biological membrane withshort pulses of electricity to form a micropore of up to about 1000 μmin diameter.

[0034] The invention further comprises the method of enhancing thetransmembrane flux rate of a permeant wherein said permeant comprises anucleic acid. More specifically, the invention includes the methodwherein said nucleic acid comprises DNA or wherein the nucleic acidcomprises RNA.

[0035] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant wherein the micropore in thebiological membrane extends into a portion of the outer layer of thebiological membrane ranging from 1 to 30 microns in depth, extendsthrough the outer layer of the biological membrane ranging from 10 to200 microns in depth, extends into the connective tissue layer of thebiological membrane ranging from 100 to 5000 microns in depth, orextends through the connective tissue layer of the biological membraneranging from 1000 to 10000 microns in depth.

[0036] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant, wherein the micropore penetratesthe biological membrane to a depth determined to facilitate desiredactivity of the selected permeant.

[0037] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant wherein the permeant comprises apolypeptide, including wherein the polypeptide is a protein or apeptide, and further including wherein the peptide comprises insulin ora releasing factor; a carbohydrate, including wherein the carbohydratecomprises a heparin; an analgesic, including wherein the analgesiccomprises an opiate; a vaccine; or a steroid.

[0038] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant wherein the permeant is associatedwith a carrier. The invention further includes the method wherein thecarrier comprises liposomes; lipid complexes; microparticles; orpolyethylene glycol compounds. More specifically, the invention furtherincludes the method wherein the permeant is a vaccine in combinationwith the method wherein the permeant is associated with a carrier.

[0039] The invention further includes the method of enhancing thetransmembrane flux rate of a permeant wherein the permeant comprises asubstance which has the ability to change its detectable response to astimulus when in the proximity of an analyte present in the organism.

[0040] An object of the invention is to provide a method for controllingtransmembrane flux rates of drugs or other molecules into the body and,if desired, into the bloodstream through minute perforations in thebiological membrane, including stratum corneum or other layers of theskin or in the mucosa or outer layers of a plant.

[0041] It is still another object of the invention to provide a methodof delivering drugs into the body through micropores in the biologicalmembrane in combination with sonic energy, permeation enhancers,pressure gradients, electromagnetic energy, thermal energy, and thelike.

[0042] An object of the invention is to minimize the barrier propertiesof the biological membrane using poration to controllably collectanalytes from within the body through perforations in the biologicalmembrane to enable the monitoring of these analytes.

[0043] It is also an object of the invention to provide a method ofmonitoring selected analytes in the body through micropores in thebiological membrane in combination with sonic energy, permeationenhancers, pressure gradients, electromagnetic energy, mechanicalenergy, thermal energy, and the like.

[0044] These and other objects may be accomplished by providing a methodfor monitoring the concentration of an analyte in an individual's bodycomprising the steps of enhancing the permeability of the biologicalmembrane of a selected area of the individual's body surface to theanalyte by means of

[0045] (a) porating the biological membrane of the selected area bymeans that form a micropore in the biological membrane optionallywithout causing serious damage to the underlying tissues, therebyreducing the barrier properties of the biological membrane to thewithdrawal of the analyte;

[0046] (b) collecting a selected amount of the analyte; and

[0047] (c) quantitating the analyte collected.

[0048] In one preferred embodiment, the method further comprisesapplying sonic energy to the porated selected area at a frequency in therange of about 5 kHz to 100 MHz, wherein the sonic energy is modulatedby means of a member selected from the group consisting of frequencymodulation, amplitude modulation, phase modulation, and combinationsthereof. In another preferred embodiment, the method comprisescontacting the selected area of the individual's body with a chemicalenhancer with the application of electromagnetic, thermal, mechanical,or sonic energy to further enhance analyte withdrawal.

[0049] Porating of the biological membrane is accomplished by meansselected from the group consisting of (a) ablating the biologicalmembrane by contacting a selected area, up to about 1000 μm across, ofthe biological membrane with a heat source such that the temperature oftissue-bound water and other vaporizable substances in the selected areais elevated above the vaporization point of the water and othervaporizable substances thereby removing the biological membrane in theselected area; (b) puncturing the biological membrane with amicro-lancet calibrated to form a micropore of up to about 1000 μm indiameter; (c) ablating the biological membrane by focusing a tightlyfocused beam of sonic energy onto the stratum corneum; (d) hydraulicallypuncturing the biological membrane with a high pressure jet of fluid toform a micropore of up to about 1000 μm in diameter and (e) puncturingthe biological membrane with short pulses of electricity to form amicropore of up to about 1000 μm in diameter.

[0050] One preferred embodiment of thermally ablating the biologicalmembrane comprises treating at least the selected area with an effectiveamount of a dye that exhibits strong absorption over the emission rangeof a pulsed light source and focusing the output of a series of pulsesfrom the pulsed light source onto the dye such that the dye is heatedsufficiently to conductively transfer heat to the stratum corneum toelevate the temperature of tissue-bound water and other vaporizablesubstances in the selected area above the vaporization point of thewater and other vaporizable substances. Preferably, the pulsed lightsource emits at a wavelength that is not significantly absorbed by skin.For example, the pulsed light source can be a laser diode emitting inthe range of about 630 to 1550 nm, a laser diode pumped opticalparametric oscillator emitting in the range of about 700 and 3000 nm, ora member selected from the group consisting of arc lamps, incandescentlamps, and light emitting diodes. A sensing system for determining whenthe barrier properties of the stratum corneum have been surmounted canalso be provided. One preferred sensing system comprises lightcollection means for receiving light reflected from the selected areaand focusing the reflected light on a photodiode, a photodiode forreceiving the focused light and sending a signal to a controller whereinthe signal indicates a quality of the reflected light, and a controllercoupled to the photodiode and to the pulsed light source for receivingthe signal and for shutting off the pulsed light source when apreselected signal is received.

[0051] In another preferred embodiment, the method further comprisescooling the selected area of biological membrane and adjacent tissueswith cooling means such that said selected area and adjacent tissues arein a selected cooled, steady state, condition prior to, during, and/orafter poration.

[0052] In still another preferred embodiment, the method comprisesablating the biological membrane such that interstitial fluid exudesfrom the micropores, collecting the interstitial fluid, and analyzingthe analyte in the collected interstitial fluid. After the interstitialfluid is collected, the micropore can be sealed by applying an effectiveamount of energy from the laser diode or other light source such thatinterstitial fluid remaining in the micropore is caused to coagulate.Preferably, vacuum is applied to the porated selected area to enhancecollection of interstitial fluid.

[0053] In yet another preferred embodiment, the method comprises, priorto porating the biological membrane, illuminating at least the selectedarea with light such that the selected area illuminated with the lightis sterilized.

[0054] Another preferred method of porating the biological membranecomprises contacting the selected area with a solid element such thatthe temperature of the selected area is raised from ambient temperatureto greater than 100° C. within about 10 nanoseconds to 50 ms and thenreturning the temperature of the selected area to approximately ambientskin temperature within about 1 to 50 ms, wherein this cycle of raisingthe temperature and returning to approximately ambient temperature isrepeated a number of time effective for reducing the barrier propertiesof the biological membrane. Preferably, the step of returning toapproximately ambient temperature is carried out by withdrawing thesolid element from contact with the biological membrane. It is alsopreferred to provide means for monitoring electrical impedance betweenthe solid element and the body through the selected area of biologicalmembrane and adjacent tissues and means for advancing the position ofthe solid element such that as the ablation occurs with a concomitantreduction in resistance, the advancing means advances the solid elementsuch that the solid element is in contact with the biological membraneduring heating of the solid element. Further, it is also preferred toprovide means for withdrawing the solid element from contact with thebiological membrane, wherein the monitoring means is capable ofdetecting a change in impedance associated with contacting a layerunderlying the biological membrane or a layer thereof and sending asignal to the withdrawing means to withdrawn the solid element fromcontact with the biological membrane. The solid element can be heated byan ohmic heating element, can have a current loop having a highresistance point wherein the temperature of the high resistance point ismodulated by passing a modulated electrical current through said currentloop to effect the heating, or can be positioned in a modulatablealternating magnetic field of an excitation coil such that energizingthe excitation coil with alternating current produces eddy currentssufficient to heat the solid element by internal ohmic losses.

[0055] A method for enhancing the transmembrane flux rate of an activepermeant into a selected area of a body comprising the steps ofenhancing the permeability of the biological membrane layer of theselected area of the body surface to the active permeant by means of

[0056] (a) porating the biological membrane of the selected area bymeans that form a micropore in the biological membrane optionallywithout causing serious damage to the underlying tissues and therebyreducing the barrier properties of the biological membrane to the fluxof the active permeant; and

[0057] (b) contacting the porated selected area with a compositioncomprising an effective amount of the permeant such that the flux of thepermeant into the body is enhanced.

[0058] In a preferred embodiment, the method further comprises applyingenergy to the porated selected area for a time and at an intensity and afrequency effective to create a fluid streaming effect and therebyenhance the transmembrane flux rate of the permeant into the body.

[0059] A method is also provided for applying a tattoo to a selectedarea of skin on an individual's body surface comprising the steps of:

[0060] (a) porating the stratum corneum of the selected area by meansthat form a micropore in the stratum corneum optionally without causingserious damage to the underlying tissues and thereby reduce the barrierproperties of the stratum corneum to the flux of a permeant; and

[0061] (b) contacting the porated selected area with a compositioncomprising an effective amount of a tattooing ink as a permeant suchthat the flux of said ink into the body is enhanced.

[0062] A method is still further provided for reducing a temporal delayin diffusion of an analyte from blood of an individual to saidindividual's interstitial fluid in a selected area of biologicalmembrane comprising applying means for cooling to said selected area ofskin.

[0063] A method is yet further provided for reducing evaporation ofinterstitial fluid and the vapor pressure thereof, wherein saidinterstitial fluid is being collected from a micropore in a selectedarea of the biological membrane of an individual, comprising applyingmeans for cooling to said selected area of biological membrane.

[0064] In accordance with still further embodiments, the presentinvention is directed to a method for delivering bioactive agents intothe body through micropores formed at selected depths in a biologicalmembrane, such as the skin or mucous membrane or outer layer of a plant.The method involves porating an outer layer of the biological membranethrough any of the poration techniques known in the art, but to asufficient and desired depth into or through the biological membrane,and contacting the porated site with an effective quantity of thebioactive agent of low or high molecular weight and size. This processcan be enhanced by applying further permeation enhancement measureseither before, during or after the bioactive agent is delivered. Forexample, sonic energy, iontophoresis, magnetic fields, electroporation,chemical permeation enhancer, osmotic pressure and atmospheric pressuremeasures may be applied to the porated site to enhance the permeabilityof layers beneath the outer layer of the biological membrane.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0065]FIG. 1 shows a schematic representation of a system for deliveringlaser diode light and monitoring the progress of poration.

[0066]FIG. 2 shows a schematic representation of a closed-loop feedbacksystem for monitoring poration.

[0067]FIG. 3A shows a schematic representation of an optical porationsystem comprising a cooling device.

[0068]FIG. 3B shows a top view of a schematic representation of anillustrative cooling device according to FIG. 3B.

[0069]FIG. 4 shows a schematic representation of an ohmic heating devicewith a mechanical actuator.

[0070]FIG. 5 shows a schematic representation of a high resistancecurrent loop heating device.

[0071]FIG. 6 shows a schematic representation of a device for modulatingheating using inductive heating.

[0072]FIG. 7 shows a schematic representation of a closed loop impedancemonitor using changes in impedance to determine the extent of poration.

[0073] FIGS. 8A-D show cross sections of human skin treated with copperphthalocyanine and then subjected, respectively, to 0, 1, 5, and 50pulses of 810 nm light with an energy density of 4000 J/cm² for a pulseperiod of 20 ms.

[0074] FIGS. 9-11 show graphic representations of temperaturedistribution during simulated thermal poration events using opticalporation.

[0075]FIGS. 12 and 13 show graphic representations of temperature as afunction of time in the stratum corneum and viable epidermis,respectively, during simulated thermal poration events using opticalporation.

[0076] FIGS. 14-16 show graphic representations of temperaturedistribution, temperature as a function of time in the stratum corneum,and temperature as a function of time in the viable epidermis,respectively, during simulated thermal poration events using opticalporation wherein the tissue was cooled prior to poration.

[0077] FIGS. 17-19 show graphic representations of temperaturedistribution, temperature as a function of time in the stratum corneum,and temperature as a function of time in the viable epidermis,respectively, during simulated thermal poration events wherein thetissue was heated with a hot wire.

[0078] FIGS. 20-22 show graphic representations of temperaturedistribution, temperature as a function of time in the stratum corneum,and temperature as a function of time in the viable epidermis,respectively, during simulated thermal poration events wherein thetissue was heated with a hot wire and the tissue was cooled prior toporation.

[0079]FIGS. 23 and 24 show graphic representations of temperaturedistribution and temperature as a function of time in the stratumcorneum, respectively, during simulated thermal poration events whereinthe tissue is heated optically according to the operating parameters ofTankovich '803.

[0080]FIG. 25 shows a graphic representation of interstitial fluid(ISF;) and blood (*) glucose levels as a function of time.

[0081]FIG. 26 shows a scatter plot representation of the difference termbetween the ISF glucose and the blood glucose data of FIG. 25.

[0082]FIG. 27 shows a histogram of the relative deviation of the ISF tothe blood glucose levels from FIG. 25.

[0083]FIG. 28 shows a cross section of an illustrative deliveryapparatus for delivering a drug to a selected area on an individual'sskin.

[0084] FIGS. 29A-C show graphic representations of areas of skinaffected by delivery of lidocaine to selected areas where the stratumcorneum is porated (FIGS. 29A-B) or not porated (FIG. 29C).

[0085]FIG. 30 shows a plot comparing the amount of interstitial fluidharvested from micropores with suction alone ( ) and with a combinationof suction and ultrasound (*).

[0086]FIGS. 31, 32, and 33 show a perspective view of an ultrasonictransducer/vacuum apparatus for harvesting interstitial fluid, a crosssection view of the same apparatus, and cross sectional schematic viewof the same apparatus, respectively.

[0087] FIGS. 34A-B show a top view of a handheld ultrasonic transducerand a side view of the spatulate end thereof, respectively.

[0088]FIG. 35 is a graphical representation showing the enhancingeffects of microporation in the transdermal delivery of testosterone.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

[0089] It is to be understood that this invention is not limited to theparticular configurations, process steps, and materials disclosed hereinas such configurations, process steps, and materials may vary somewhat.It is also to be understood that the terminology employed herein is usedfor the purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

[0090] It must be noted that, as used herein the singular forms “a,”“an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to a method fordelivery of “a drug” includes reference to delivery of a mixture of twoor more drugs, reference to “an analyte” includes reference to one ormore of such analytes, and reference to “a permeation enhancer” includesreference to a mixture of two or more permeation enhancers or techniquessuch as a combination of ultrasound and electroporation.

[0091] Thus, as used herein, the singular form may be usedinterchangeably with the plural form, and vice versa, i.e.: “layer”could mean layers or “layers” could mean layer.

[0092] As used herein, “including” or “includes” or the like meansincluding, without limitation.

[0093] In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

[0094] As used herein “organism” means the entire animal or plant beingacted upon by the methods described herein.

[0095] As used herein, “poration,” “microporation,” or any such similarterm means the formation of a small hole or pore to a desired depth inor through the biological membrane, such as skin or mucous membrane, orthe outer layer of an organism to lessen the barrier properties of thisbiological membrane to the passage of analytes from below the surfacefor analysis or the passage of permeants or drugs into the body forselected purposes, or for certain medical or surgical procedures. Themicroporation process referred to herein is distinguished from theopenings formed by electroporation principally by the minimum dimensionsof the micropores which shall be no smaller than 1 micron across and atleast 1 micron in depth, whereas the openings formed withelectroporation are typically only a few nanometers in any dimension.Nevertheless, electroporation is useful to facilitate uptake of selectedpermeants by the targeted tissues beneath the outer layers of anorganism after the permeant has passed through the micropores into thesedeeper layers of tissue. Preferably the hole or micropore will be nolarger than about 1 mm in diameter, and more preferably no larger thanabout 300 μm in diameter, and will extend to a selected depth, asdescribed hereinafter.

[0096] As used herein, “micropore” or “pore” means an opening formed bythe microporation method.

[0097] As used herein “ablation” means the controlled removal ofmaterial which may include cells or other components comprising someportion of a biological membrane or tissue caused by any of thefollowing: kinetic energy released when some or all of the vaporizablecomponents of such material have been heated to the point thatvaporization occurs and the resulting rapid expansion of volume due tothis phase change causes this material, and possibly some adjacentmaterial, to be removed from the ablation site; thermal, mechanical, orsonic decomposition of some or all off the tissue at the poration site.

[0098] As used herein ablation of a tissue or puncture of a tissue maybe achieved utilizing the same energy source.

[0099] As used herein, “tissue” means any component of an organismincluding but not limited to, cells, biological membranes, bone,collagen, fluids and the like comprising some portion of the organism.

[0100] As used herein, “sonic” or “acoustic” are interchangeable andcover the frequency space from 0.01 Hz and up.

[0101] As used herein, “ultrasonic” describes a subset of soniccomprising frequencies greater or equal to 20,000 Hz with no upperlimit.

[0102] As used herein “puncture” or “micro-puncture” means the use ofmechanical, hydraulic, sonic, electromagnetic, or thermal means toperforate wholly or partially a biological membrane such as the skin ormucosal layers of an animal or the outer tissue layers of a plant.

[0103] To the extent that “ablation” and “puncture” accomplish the samepurpose of poration, i.e. the creating a hole or pore in the biologicalmembrane optionally without significant damage to the underlyingtissues, these terms may be used interchangeably.

[0104] As used herein, “penetration enhancement” or “permeationenhancement” means an increase in the permeability by utilization of apermeation enhancer of a biological membrane such as the skin or mucosalor buccal membrane or a plant's outer layer of tissue to a bioactiveagent, drug, analyte, dye, stain, microparticle, microsphere, compound,or other chemical formulation (also called “permeant”), i.e., so as toincrease the rate at which a bioactive agent, drug, analyte, stain,micro-particle, microsphere, compound, or other chemical formulationpermeates the biological membrane and facilitates the withdrawal ofanalytes out through the biological membrane or the delivery ofsubstances through the biological membrane and into the underlyingtissues. The enhanced permeation effected through the use of suchenhancers can be observed, for example, by observing diffusion of a dye,as a permeant, through animal or human skin using a diffusion apparatus.

[0105] As used herein, “penetration enhancer,” “permeation enhancer,”“enhancer,” and the like includes all substances and techniques thatincrease the flux of a permeant, analyte, or other molecule across theskin, and is limited only by functionality. In other words, all cellenvelope disordering compounds and solvents and physical techniques suchas electroporation, iontophoresis, magnetic fields, sonic energy,thermal energy, or mechanical pressure or manipulation such as a localmassaging of the site and any chemical enhancement agents are intendedto be included.

[0106] As used herein “chemical enhancer” means a substance thatincreases the flux of a permeant or analyte or other substance across abiological membrane and is limited only by function.

[0107] As used herein, “dye,” “stain,” and the like shall be usedinterchangeably and refer to a biologically suitable chromophore thatexhibits suitable absorption over some or all of the emission range of apulsed light source used to ablate tissues to form micropores therein.

[0108] As used herein, “transdermal” or “percutaneous” or“transmembrane” or “transmucosal” or “transbuccal” means passage of apermeant into or through the biological membrane or tissue to achieveeffective therapeutic blood levels or tissue levels of a drug, or thepassage of a molecule present in the body (“analyte”) out through thebiological membrane or tissue so that the analyte molecule may becollected on the outside of the body.

[0109] As used herein, the term “bioactive agent,” “permeant,” “drug,”or “pharmacologically active agent” or “deliverable substance” or anyother similar term means any chemical or biological material or compoundsuitable for delivery by the methods previously known in the art and/orby the methods taught in the present invention, that induces a desiredeffect, such as a biological or pharmacological effect, which mayinclude but is not limited to (1) having a prophylactic effect on theorganism and preventing an undesired biological effect such aspreventing an infection, (2) alleviating a condition caused by adisease, for example, alleviating pain or inflammation caused as aresult of disease, (3) either alleviating, reducing, or completelyeliminating the disease from the organism, and/or (4) the placementwithin the viable tissue layers of the organism of a compound orformulation which can react, optionally in a reversible manner, tochanges in the concentration of a particular analyte and in so doingcause a detectable shift in this compound or formulation's measurableresponse to the application of energy to this area which may beelectromagnetic, mechanical or acoustic. The effect may be local, suchas providing for a local anesthetic effect, or it may be systemic. Thisinvention is not drawn to novel permeants or to new classes of activeagents other than by virtue of the microporation technique, althoughsubstances not typically being used for transdermal, transmucosal,transmembrane or transbuccal delivery may now be useable. Rather it isdirected to the mode of delivery of bioactive agents or permeants thatexist in the art or that may later be established as active agents andthat are suitable for delivery by the present invention.

[0110] Such substances include broad classes of compounds normallydelivered into the organism, including through body surfaces andmembranes, including skin as well as by injection, including needle,hydraulic, or hypervelocity methods. In general, this includes but isnot limited to: Polypeptides, including proteins and peptides (e.g.,insulin); releasing factors, including Luteinizing Hormone ReleasingHormone (LHRH); carbohydrates (e.g., heparin); nucleic acids; vaccines;and pharmacologically active agents such as antiinfectives such asantibiotics and antiviral agents; analgesics and analgesic combinations;anorexics; antihelminthics; antiarthritics; antiasthmatic agents;anticonvulsants; antidepressants; antidiabetic agents; antidiarrheals;antihistamines; antiinflammatory agents; antimigraine preparations;antinauseants; antineoplastics; antiparkinsonism drugs; antipruritics;antipsychotics; antipyretics; antispasmodics; anticholinergics;sympathomimetics; xanthine derivatives; cardiovascular preparationsincluding potassium and calcium channel blockers, beta-blockers,alpha-blockers, and antiarrhythmics; antihypertensives; diuretics andantidiuretics; vasodilators including general coronary, peripheral andcerebral; central nervous system stimulants; vasoconstrictors; cough andcold preparations, including decongestants; hormones such as estradiol,testosterone, progesterone and other steroids and derivatives andanalogs, including corticosteroids; hypnotics; immunosuppressives;muscle relaxants; parasympatholytics; psychostimulants; sedatives; andtranquilizers. By the method of the present invention, both ionized andnonionized permeants may be delivered, as can permeants of any molecularweight including substances with molecular weights ranging from lessthan 50 Daltons to greater than 1,000,000 Daltons.

[0111] As used herein, an “effective” amount of a permeant means asufficient amount of a compound to provide the desired local or systemiceffect and performance at a reasonable benefit/risk ratio attending anytreatment. An “effective” amount of an enhancer as used herein means anamount selected so as to provide the desired increase in tissuepermeability and the desired depth of penetration, rate ofadministration, and amount of 15 permeant delivered.

[0112] As used herein, “carriers” or “vehicles” refer to carriermaterials without significant pharmacological activity at the quantitiesused that are suitable for administration with other permeants, andinclude any such materials known in the art, e.g., any liquid, gel,solvent, liquid diluent, solubilizer, microspheres, liposomes,microparticles, lipid complexes, or the like, that is sufficientlynontoxic at the quantities employed and does not interact with the drugto be administered in a deleterious manner. Examples of suitablecarriers for use herein include water, buffers, mineral oil, silicone,inorganic or organic gels, aqueous emulsions, liquid sugars, lipids,microparticles, waxes, petroleum jelly, and a variety of other oils andpolymeric materials.

[0113] As used herein, a “biological membrane” means a tissue materialpresent within a living organism that separates one area of the organismfrom another and, in many instances, that separates the organism fromits outer environment. Skin and mucous and buccal membranes are thusincluded as well as the outer layers of a plant. Also, the walls of acell or a blood vessel would be included within this definition.

[0114] As used herein, “mucous membrane” or “mucosa” refers to theepithelial linings of the mouth, nasopharynx, throat, respiratory tract,urogenital tract, anus, eye, gut and all other surfaces accessible viaan endoscopic device such as the bladder, colon, lung, blood vessels,heart and the like.

[0115] As used herein, the “buccal membrane” includes the mucousmembrane of the mouth.

[0116] As used herein, “outer layer” and “connective-tissue layer” areparts of the biological membrane and have the following meanings. “Outerlayer” means all or part of the. epidermis of the skin or the epitheliallining of the mucous membrane or the outer layer of a plant. The mostsuperficial portion of the animal epidermis is the stratum corneum, asis well known in the art. The deeper portion of the epidermis is called,for simplicity, the “viable cell layer” hereinafter. Beneath the outerlayer is the “connective tissue layer.”

[0117] The connective tissue layer means the dermis in the skin or thelamina propria in the mucous membrane or other underlying tissues inplants or animals.

[0118] As used herein, “organism” or “individual” or “subject” or “body”refers to any of a human, animal, or plant to which the presentinvention may be applied.

[0119] As used herein, “analyte” means any chemical or biologicalmaterial or compound suitable for passage through a biological membraneby the technology taught in this present invention, or by technologypreviously known in the art, of which an individual might want to knowthe concentration or activity inside the body. Glucose is a specificexample of an analyte because it is a sugar suitable for passage throughthe skin, and individuals, for example those having diabetes, might wantto know their blood glucose levels. Other examples of analytes include,but are not limited to, such compounds as sodium, potassium, bilirubin,urea, ammonia, calcium, lead, iron, lithium, salicylates, antibodies,hormones, or an exogenously delivered substance and the like.

[0120] As used herein, “into” or “in” a biological membrane or layerthereof includes penetration in or through only one or more layers(e.g., all or part of the stratum corneum or the entire outer layer ofthe skin or portion thereof).

[0121] As used herein, “through” a biological membrane or layer thereofmeans through the entire depth of the biological membrane or layerthereof.

[0122] As used herein, “transdermal flux rate” is the rate of passage ofany analyte out through the skin of a subject or the rate of passage ofany bioactive agent, drug, pharmacologically active agent, dye, particleor pigment in and through the skin separating the organism from itsouter environment. “Transmucosal flux rate” and “transbuccal flux rate”refer to such passage through mucosa and buccal membranes and“transmembrane flux rate” refers to such passage through any biologicalmembrane.

[0123] As used herein, “transdermal,” “transmucosal,” “transbuccal” and“transmembrane” may be used interchangeably as appropriate within thecontext of their use.

[0124] As used herein, the terms “intensity amplitude,” “intensity,” and“amplitude” are used synonymously and refer to the amount of energybeing produced by a sonic, thermal, mechanical or electromagnetic energysystem.

[0125] As used herein, “frequency modulation” or “sweep” means acontinuous, graded or stepped variation in the frequency of a sonic,thermal, mechanical or electromagnetic energy in a given time period. Afrequency modulation is a graded or stepped variation in frequency in agiven time period, for example 5.4-5.76 MHz in 1 sec., or 5-10 MHz in0.1 sec., or 10-5 MHz in 0.1 sec., or any other frequency range or timeperiod that is appropriate to a specific application. A complexmodulation can include varying both the frequency and intensitysimultaneously. For example, FIGS. 4A and 4B of U.S. Pat. No. 5,458,140could, respectively, represent amplitude and frequency modulations beingapplied simultaneously to a single sonic energy transducer.

[0126] As used herein, “amplitude modulation” means a continuous, gradedor stepped variation in the amplitude or intensity of a sonic, thermal,mechanical or electromagnetic energy in a given time period.

[0127] As used herein “phase modulation” means the timing of a sonic,thermal, mechanical or electromagnetic energy or signal has been changedrelative to its initial state. An example is shown in FIG. 4C of U.S.Pat. No. 5,458,140. The frequency and amplitude of the signal can remainthe same. A phase modulation can be implemented with a variable delaysuch as to selectively retard or advance the signal temporarily inreference to its previous state, or to another signal.

[0128] As used herein “signal,” or “energy” may be used synonymously.The sonic, thermal, mechanical or electromagnetic energy, in its variousapplications such as with frequency, intensity or phase modulation, orcombinations thereof and the use of chemical enhancers combined withsonic, thermal, mechanical or electromagnetic energy, as describedherein, can vary over a frequency range of between about 0.01 Hz to 1000MHz, with a range of between about 0.1 Hz and 30 MHz being preferred.

[0129] As used herein, “non-invasive” means not requiring the entry of aneedle, catheter, or other invasive instrument into a part of thesubject including the skin or a mucous membrane.

[0130] As used herein, “minimally invasive and “non-invasive” aresynonymous.

[0131] As used herein, “microparticles” or “microspheres” or“nanoparticles” or “nanospheres” or “liposomes” or “lipid complexes” maybe used interchangeably.

[0132] Means for Poration of the Biological Membrane

[0133] The formation of a micropore in the biological membrane can beaccomplished by various state of the art means as well as certain meansdisclosed herein that are improvements thereof. While the followingtechniques and examples are made with respect to porating the biologicalmembrane, it should be understood that the improvements described hereinalso apply to porating the mucous or buccal membrane or the outer layersof a plant.

[0134] The use of laser ablation as described by Jacques et al. in U.S.Pat. No. 4,775,361 and by Lane et al., supra, certainly provide onemeans for ablating the stratum corneum using an excimer laser. At 193 nmwavelength, and 14 ns pulse width, it was found that about 0.24 to 2.8μm of stratum corneum could be removed by each laser pulse at radiantexposure of between about 70 and 480 mJ/cm². As the pulse energyincreases, more tissue is removed from the stratum corneum and fewerpulses are required for complete poration of this layer. The lowerthreshold of radiant exposure that must be absorbed by the stratumcorneum within the limit of the thermal relaxation time to causesuitable micro-explosions that result in tissue ablation is about 70mJ/cm² within a 50 millisecond (ms) time. In other words, a total of 70mJ/cm² must be delivered within a 50 ms window. This can be done in asingle pulse of 70 mJ/cm² or in 10 pulses of 7 mJ/cm², or with acontinuous illumination of 1.4 watts/cm² during the 50 ms time. Theupper limit of radiant exposure is that which will ablate the stratumcorneum without damage to underlying tissue and can be empiricallydetermined from the light source, wavelength of light, and othervariables that are within the experience and knowledge of one skilled inthis art.

[0135] By “delivery”, in the context of the application of energy, ismeant that the stated amount of energy is absorbed by the tissue to beablated. At the excimer laser wavelength of 193 nm, essentially 100%absorption occurs within the first 1 or 2 μm of stratum corneum tissue.Assuming the stratum corneum is about 20 μm thick, at longerwavelengths, such as 670 nm, only about 5% of incident light is absorbedwithin the 20 μm layer. This means that about 95% of the high power beampasses into the tissues underlying the stratum corneum where it willlikely cause significant damage. In the context of delivery of abioactive agent, the term means providing the bioactive agent to thedesired location.

[0136] The ideal is to use only as much power as is necessary toperforate the biological membrane or other selected skin, mucosal, ortissue layers without causing bleeding, thermal, or other unacceptabledamage to underlying and adjacent tissues from which analytes are to beextracted or permeants delivered.

[0137] It would be beneficial to use sources of energy more economicalthan energy from excimer lasers. Excimer lasers, which emit light atwavelengths in the far UV region, are much more expensive to operate andmaintain than, for example, diode lasers that emit light at wavelengthsin visible and IR regions (600 to 1800 nm). However, at the longerwavelengths, the biological membrane becomes increasingly moretransparent and absorption occurs primarily in the underlying tissues.

[0138] The present invention facilitates a rapid and minimally traumaticmethod of eliminating the barrier function of the biological membrane tofacilitate the transmembrane transport of substances into the body whenapplied topically or to access the analytes within the body foranalysis. The method utilizes a procedure which begins with the contactapplication of a small area heat source to the targeted area of thebiological membrane.

[0139] The heat source must have several important properties, as willnow be described. First, the heat source must be sized such that contactwith the biological membrane is confined to a small area, typicallyabout 1 to 1000 μm in diameter. Second, it must have the capability tomodulate the temperature of the biological membrane at the contact pointfrom ambient surface temperature to greater than the vaporization pointof a sufficient amount of the components within the biological membraneand then return to approximately ambient temperature with cycle times tominimize collateral damage to viable tissues and trauma to the subject.This modulation can be created electronically, mechanically, orchemically.

[0140] Additionally, for selected applications, an inherent depthlimiting feature of the microporation process can be facilitated if theheat source has both a small enough thermal mass and limited energysource to elevate its temperature such that when it is placed in contactwith tissues with more than 30% water content, the thermal dispersion inthese tissues is sufficient to limit the maximum temperature of the heatsource to less than 100 C. This feature effectively stops the thermalvaporization process once the heat probe had penetrated through thestratum corneum into or through the lower layers of the epidermis.

[0141] However, if one utilizes a heat probe which can continue todeliver sufficient energy into or through the hydrated viable tissuelayers beneath the outer layer of the biological membrane, the porationprocess can continue into the body to a selected depth, penetratingthrough deeper layers including, e.g., in the case of the skin, throughthe epidermis, the dermis, and into the subcutaneous layers below ifdesired. The concern when a system is designed to create a microporeextending some distance into or through the viable tissues beneath thestratum corneum, mucosal or buccal membranes is principally how tominimize damage to the adjacent tissue and the sensation to the subjectduring the poration process. Experimentally, we have shown that if theheat probe used is a solid, electrically or optically heated element,with the active heated probe tip physically defined to be no more than afew hundred microns across and protruding up to a few millimeters fromthe supporting base, that a single pulse, or multiple pulses of currentcan deliver enough thermal energy into or through the tissue to allowthe ablation to penetrate as deep as the physical design allows, forexample, until the support base acts as a component to limit the extentof the penetration into or through the tissue, essentially restrictingthe depth to which the heat probe can penetrate into a micropore tocontact fresh, unporated tissue. If the electrical and thermalproperties of said heat probe, when it is in contact with the tissues,allow the energy pulse to modulate the temperature of said probe rapidlyenough, this type of deep tissue poration can be accomplished withessentially no pain to the subject. Experiments have shown that if therequired amount of thermal energy is delivered to the probe within lessthan roughly 20 milliseconds, that the procedure is painless.Conversely, if the energy pulse must be extended beyond roughly 20milliseconds, the sensation to the subject increases rapidly andnon-linearly as the pulse width is extended.

[0142] An electrically heated probe design which supports this type ofselected depth poration can be built by bending a 50 to 150 microndiameter tungsten wire into a sharp kink, forming a close to 180 degreebend with a minimal internal radius at this point. This miniature ‘V’shaped piece of wire can then be mounted such that the point of the ‘V’extends some distance out from a support piece which has copperelectrodes deposited upon it. The distance to which the wire extends outfrom the support will define the maximum penetration distance into orthrough the tissue when the wire is heated. Each leg of the tungsten ‘V’will be attached to one of the electrodes on the support carrier whichin turn can be connected to the current pulsing circuit. When thecurrent is delivered to the wire in an appropriately controlled fashion,the wire will rapidly heat up to the desired temperature to effect thethermal ablation process in a single pulse or in multiple pulses ofcurrent. By monitoring the dynamic impedance of the probe and knowingthe coefficient of resistance versus temperature of the tungstenelement, closed loop control of the temperature of the contact point caneasily be established. Also, by dynamically monitoring the impedancethrough the body from the contact point of the probe and a secondelectrode placed some distance away, the depth of the pore can beestimated based on the different impedance properties of the tissue asone penetrates deeper into the body.

[0143] An optically heated probe design which supports this type ofselected depth poration can be built by taking an optical fiber andplacing on one end a tip comprised of a solid cap or coating. A lightsource such as a laser diode will be coupled into the other end of thefiber. The side of tip facing the fiber must have a high enoughabsorption coefficient over the range of wavelengths emitted by thelight source that when the photons reach the IS end of the fiber andstrike this face, some of them will be absorbed and subsequently causethe tip to heat up. The specific design of this tip, fiber and sourceassembly may vary widely, however fibers with gross diameters of 50 to1000 microns across are common place items today and sources emitting upto thousands of watts of optical energy are similarly common place. Thetip forming the actual heat probe can be fabricated from a high meltingpoint material, such as tungsten and attached to the fiber by machiningit to allow the insertion of the fiber into a cylindrical bore at thefiber end. If the distal end of the tip has been fabricated to limit thethermal diffusion away from this tip and back up the supporting cylinderattaching the tip to the fiber within the time frame of the opticalpulse widths used, the photons incident upon this tip will elevate thetemperature rapidly on both the fiber side and the contact side which isplaced against the tissues surface. The positioning of the fiber/tipassembly onto the tissue surface, can be accomplished with a simplemechanism designed to hold the tip against the surface under some springtension such that as the tissue beneath it is ablated, the tip itselfwill advance into the tissue. This allows the thermal ablation processto continue into or through the tissue as far as one desires. Anadditional feature of this optically heated probe design is that bymonitoring the black body radiated energy from the heated tip that iscollected by the fiber, a very simple closed loop control of the tiptemperature can be effected. Also, as described earlier, by dynamicallymonitoring the impedance through the body from the contact point of theprobe and a second electrode placed some distance away, the depth of thepore can be determined based on the different impedance properties ofthe tissue as one penetrates deeper into the body. The relationshipbetween pulse width and sensation for this design is essentially thesame as for the electrically heated probe described earlier.

[0144] Impedance can be used to determine the depth of a pore made byany means. It can be used as an input to a control system for makingpores of selected depth. The impedance measured may be the compleximpedance measured at a frequency selected to highlight the impedanceproperties of the selected tissues in a selected organism.

[0145] An additional feature of this invention is the large increase inefficiency which can be gained by combining the poration of the outerlayers of the biological membrane with other permeation enhancementtechniques which can now be optimized to function on the variousbarriers to effective delivery of the desired compound into or throughthe internal spaces it needs to go to be bio-effective. In particular,if one is delivering a DNA compound either naked, fragmented,encapsulated or coupled to another agent, it is often desired to get theDNA into the living cells without killing the cell to allow the desireduptake and subsequent performance of the therapy. It is well know in theart that electroporation, iontophoresis, and ultrasound can causeopenings to form, temporarily, in the cell membranes and other internaltissue membranes. By having breached the stratum corneum or mucosallayer or outer layer of a plant and if desired the epidermis and dermisor deeper into a plant, electroporation, iontophoresis, magnetic fields,and sonic energy can now be used with parameters that can be tailored toact selectively on these underlying tissue barriers. For example, forany electromagnetic or sonic energy enhancement means, the specificaction of the enhancement can be designed to focus on any part of thepore, e.g., on the bottom of the pore by the design of the focusingmeans employed such as the design of the electrodes, sonic and magneticfield forming devices and the like. Alternatively, the enhancer can befocused more generally on the entire pore or the area surrounding thepore. In the case of electroporation, where pulses exceeding 50 to 150volts are routinely used to electroporate the stratum corneum or mucosallayer, in the environment we present, pulses of only a few volts can besufficient to electroporate the cell, capillary or other membraneswithin the targeted tissue. This is principally due to the dramaticreduction in the number of insulating layers present between theelectrodes once the outer surface of the biological membrane has beenopened. Similarly, iontophoresis can be shown to be effective tomodulate the flux of a fluid media containing the DNA through themicropores with very small amounts of current due to the dramaticreduction in the physical impedance to fluid flow through these poratedlayers.

[0146] Whereas ultrasound has previously been used to accelerate thepermeation of the stratum corneum or mucosal layer, by eliminating thisbarrier via the micropores, we have created the opportunity to utilizesonic energy to permeabilize the cell, capillary or other structureswithin the targeted tissue. As in the cases of electroporation andiontophoresis, we have demonstrated that the sonic energy levels neededto effect a notable improvement in the trans-membrane flux of asubstance are much lower than when stratum corneum or mucosal layers areleft intact. The mode of operation of all of these active methods,electroporation, iontophoresis, magnetic fields, mechanical forces orultrasound, when applied solely or in combination, after the poration ofbiological membrane has been effected is most similar to the parameterstypically used in in vitro applications where single cell membranes arebeing opened up for the delivery of a substance.

[0147] With the heat source placed in contact with the surface of thebiological membrane, it is cycled through a series of one or moremodulations of temperature from an initial point of ambient temperatureto a peak temperature in excess of 123° C. and back to ambient surfacetemperature. To minimize or eliminate the animal's sensory perception ofthe microporation process, these pulses are limited in duration, and theinterpulse spacing is long enough to allow cooling of the viable tissuelayers in the biological membrane, and most particularly the innervatedtissues, to achieve a mean temperature within the innervated tissues ofless than about 45 C. These parameters are based on the thermal timeconstants of the human skin's viable epidermal tissues (roughly 30-80ms) located between the heat probe and the innervated tissue in theunderlying dermis. The result of this application of pulsed thermalenergy is that enough energy is conducted into or through the stratumcorneum within the tiny target spot that the local temperature of thisvolume of tissue is elevated sufficiently higher than the vaporizationpoint of the tissue-bound water content in the stratum corneum. As thetemperature increases above 100 C, the water content of the stratumcorneum (typically 5% to 15%) within this localized spot, is induced tovaporize and expand very rapidly, causing a vapor-driven removal ofthose corneocytes in the stratum corneum located in proximity to thisvaporization event. U.S. Pat. No. 4,775,361 teaches that a stratumcorneum temperature of 123° C. represents a threshold at which this typeof flash vaporization occurs. As subsequent pulses of thermal energy areapplied, additional layers of the stratum corneum are removed until amicropore is formed through the stratum corneum down to the next layerof the epidermis, the stratum lucidum. By limiting the duration of theheat pulse to less than one thermal time constant of the epidermis andallowing any heat energy conducted into or through the epidermis todissipate for a sufficiently long enough time, the elevation intemperature of the viable layers of the epidermis is minimal. Thisallows the entire microporation process to take place without anysensation to the subject and no damage to the underlying and surroundingtissues. If the heat probe can achieve temperatures greater than 300degrees C. some of the poration may be due to the direct thermaldecomposition of the tissue.

[0148] The present invention comprises a method for painlessly, or withlittle sensation, creating microscopic holes, i.e. micropores, fromabout 1 to 1000 μm across, in a biological membrane of an organism. Thekey to successfully implementing this method is the creation of anappropriate thermal energy source, or heat probe, which is held incontact with the biological membrane. The principle technical challengein fabricating an appropriate heat probe is designing a device that hasthe desired contact with the biological membrane and that can bethermally modulated at a sufficiently high frequency.

[0149] It is possible to fabricate an appropriate heat probe bycontacting the biological membrane with a suitable light-absorbingcompound, such as a dye or stain, or any thin film or substance selectedbecause of its ability to absorb light at the wavelength emitted by aselected light source. In this instance, the selected light source maybe a laser diode emitting at a wavelength which would not normally beabsorbed by the biological membrane. By focusing the light source to asmall spot on the surface of the topical layer of the dye, stain, thinfilm or substance the targeted area can be temperature modulated byvarying the intensity of the light flux focused on it. It is possible toutilize the energy from laser sources emitting at a longer wavelengththan an excimer laser by first topically applying to the stratum corneuma suitable light-absorbing compound, such as a dye, stain, thin film orsubstance selected because of its ability to absorb light at thewavelength emitted by the laser source. The same concept can be appliedat any wavelength and one must only choose an appropriate dye or stainand optical wavelength. One need only look to any reference manual tofind which suitable dyes and wavelength of the maximum absorbance ofthat dye. One such reference is Green, The Sigma-Aldrich Handbook ofStains, Dyes and Indicators, Aldrich Chemical Company, Inc. Milwaukee,Wis. (1991). For example, copper phthalocyanine (Pigment Blue 15; CPC)absorbs at about 800 nm; copper phthalocyanine tetrasulfonic acid (AcidBlue 249) absorbs at about 610 nm; and Indocyanine Green absorbs atabout 775 nm; and Cryptocyanine absorbs at about 703 nm. CPC isparticularly well suited for this embodiment for the following reasons:it is a very stable and inert compound, already approved by the FDA foruse as a dye in implantable sutures; it absorbs very strongly atwavelengths from 750 nm to 950 nm, which coincide well with numerous lowcost, solid state emitters such as laser diodes and LEDs, and inaddition, this area of optical bandwidth is similarly not absorbeddirectly by the skin tissues in any significant amount; CPC has a veryhigh vaporization point (>550C in a vacuum) and goes directly from asolid phase to a vapor phase with no liquid phase; CPC has a relativelylow thermal diffusivity constant, allowing the light energy focused onit to selectively heat only that area directly in the focal point withvery-little lateral spreading of the ‘hot-spot’ into the surrounding CPCthereby assisting in the spatial definition of the contact heat-probe.

[0150] The purpose of this disclosure is not to make an exhaustivelisting of suitable dyes, stains, films or substances because such maybe easily ascertained by one skilled in the art from data readilyavailable.

[0151] The same is true for any desired particular pulsed light source.For example, this method may be implemented with a mechanicallyshuttered, focused incandescent lamp as the pulsed light source. Variouscatalogs and sales literature show numerous lasers operating in the nearUV, visible and near IR range. Representative lasers are HammamatsuPhotonic Systems Model PLP-02 which operates at a power output of 2×10⁻⁸J, at a wavelength of 415 nm; Hammamatsu Photonic Systems Model PLP-05which operates at a power output of 15 J, at a wavelength of 685 nm;SDL, Inc., SDL-3250 Series pulsed laser which operates at a power outputof 2×10⁶ J at a wavelength of about 800-810 nm; SDL, Inc., ModelSDL-8630 which operates at a power output of 500 mW at a wavelength ofabout 670 nm; Uniphase Laser Model AR-081-15000 which operates at apower output of 15,000 mW at a wavelength of 790-830 nm; Toshiba AmericaElectronic Model TOLD9150 which operates at a power output of 30 mW at awavelength of 690 nm; and LiCONIX, Model Diolite 800-50 which operatesat a power 50 mW at a wavelength of 780 nm.

[0152] For purposes of the present invention a pulsed laser light sourcecan emit radiation over a wide range of wavelengths ranging from betweenabout 100 nm to 12,000 nm. Excimer lasers typically will emit over arange of between about 100 to 400 nm. Commercial excimer lasers arecurrently available with wavelengths in the range of about 193 nm to 350nm. Preferably a laser diode will have an emission range of betweenabout 380 to 1550 nm. A frequency doubled laser diode will have anemission range of between about 190 and 775 nm. Longer wavelengthsranging from between about 1300 and 3000 nm may be utilized using alaser diode pumped optical parametric oscillator. It is expected, giventhe amount of research taking place on laser technology, that theseranges will expand with time.

[0153] Delivered or absorbed energy need not be obtained from a laser asany source of light, whether it is from a laser, a short arc lamp suchas a xenon flashlamp, an incandescent lamp, a light-emitting diode(LED), the sun, or any other source may be used. Thus, the particularinstrument used for delivering electromagnetic radiation is lessimportant than the wavelength and energy associated therewith. Anysuitable instrument capable of delivering the necessary energy atsuitable wavelengths, i.e. in the range of about 100 nm to about 12,000nm, can be considered within the scope of the invention. The essentialfeature is that the energy must be absorbed by the light-absorbingcompound to cause localized heating thereof, followed by conduction ofsufficient heat to the tissue to be ablated within the time frameallowed.

[0154] In one illustrative embodiment, the heat probe itself is formedfrom a thin layer, preferably about 5 to 1000 μm thick, of a solid,non-biologically active substance placed in contact with a selected areaof an individual's skin that is large enough to cover the site where amicropore is to be created. The specific formulation of the chemicalcompound is chosen such that it exhibits high absorption over thespectral range of a light source selected for providing energy to thelight-absorbing compound. The probe can be, for example, a sheet of asolid compound, a film treated or coated with or containing a suitablelight absorbing compound, or a direct application of the light-absorbingcompound to the skin as a precipitate or as a suspension in a carrier.Regardless of the configuration of the light-absorbing heat probe, itmust exhibit a low enough lateral thermal diffusion coefficient suchthat any local elevations of temperature will remain sufficientlyspatially defined and the dominant mode of heat loss will preferably bevia direct conduction into biological membrane through the point ofcontact between the skin and the probe.

[0155] The required temperature modulation of the probe can be achievedby focusing a light source onto the probe layer and modulating theintensity of this light source. If the energy absorbed within theilluminated area is sufficiently high, it will cause the probe layerheat up. The amount of energy delivered, and subsequently both the rateof heating and peak temperature of the probe layer at the focal point,can be easily modulated by varying the pulse width and peak power of thelight source. In this embodiment, it is only the small volume of probelayer heated up by the focused, incident optical energy that forms theheat probe, additional material of this probe layer which may have beenapplied over a larger area then the actual poration site is incidental.By using a solid phase light-absorbing compound with a relatively highmelting point, such as copper phthalocyanine (CPC), which remains in itssolid phase up to a temperature of greater than 550 C, the heat probecan be quickly brought up to a temperature of several hundred degreesC., and still remain in contact with the biological membrane, allowingthis thermal energy to be conducted into or through the stratum corneum.In addition, this embodiment comprises choosing a light source with anemission spectrum where very little energy would normally be absorbed inthe tissues of the biological membrane.

[0156] Once the targeted area has the light-absorbing probe layer placedin contact to it, the heat probe is formed when the light source isactivated with the focal waist of the beam positioned to be coincidentwith the surface of the treated area. The energy density of light at thefocal waist and the amount of absorption taking place within thelight-absorbing compound are set to be sufficient to bring thetemperature of the light-absorbing compound, within the area of thesmall spot defined by the focus of the light source, to greater than123° C. within a few milliseconds. As the temperature of the heat proberises, conduction into or through the biological membrane deliversenergy into or through these tissues, elevating the local temperature ofthe biological membrane. When enough energy has been delivered into orthrough this small area of biological membrane to cause the localtemperature to be elevated above the boiling point of some of the waterand other vaporizable components contained in these tissues, a flashvaporization of this material takes place, removing some portion of thebiological membrane at this location and forming a micropore.

[0157] By turning the light source on and off, the temperature of theheat probe can be rapidly modulated and the selective ablation of thesetissues can be achieved, allowing a very precisely dimensioned hole tobe created, which can selectively penetrate only through the first 10 to30 microns of the biological membrane, or can be made deeper.

[0158] An additional feature of this embodiment is that by choosing alight source that would normally have very little energy absorbed by thebiological membrane or underlying tissues, and by designing the focusingand delivery optics to have a sufficiently high numerical aperture, thesmall amount of delivered light that does not happen to get absorbed inthe heat probe itself, quickly diverges as it penetrates deep into thebody. Since there is very little absorption at the deliveredwavelengths, essentially no energy is delivered to the biologicalmembrane directly from the light source. This three dimensional dilutionof coupled energy in the tissues due to beam divergence and the lowlevel of absorption in the untreated tissue results in a completelybenign interaction between the light beam and the tissues, with nodamage being done thereby.

[0159] In one preferred embodiment of the invention, a laser diode isused as the light source with an emission wavelength of 800±30 nm. Aheat-probe can be formed by topical application of a transparentadhesive tape that has been treated on the adhesive side with a 0.5 cmspot formed from a deposit of finely ground copper phthalocyanine (CPC).The CPC exhibits extremely high absorption coefficients in the 800 nmspectral range, typically absorbing more than 95% of the radiant energyfrom a laser diode.

[0160]FIG. 1 shows a system 10 for delivering light from such a laserdiode to a selected area of an individual's biological membrane and formonitoring the progress of the poration s process. The system comprisesa laser diode 14 coupled to a controller 18, which controls theintensity, duration, and spacing of the light pulses. The laser diodeemits a beam 22 that is directed to a collection lens or lenses 26,which focuses the beam onto a mirror 30. The beam is then reflected bythe mirror to an objective lens or lenses 34, which focuses the beam ata preselected point 38. This preselected point corresponds with theplane of an xyz stage 42 and the objective hole 46 thereof, such that aselected area of an individual's biological membrane can be irradiated.The xyz stage is connected to the controller such that the position ofthe xyz stage can be controlled. The system also comprises a monitoringsystem comprising a CCD camera 50 coupled to a monitor 54 The CCD camerais confocally aligned with the objective lens such that the progress ofthe poration process can be monitored visually on the monitor.

[0161] In another illustrative embodiment of the invention, a system ofsensing photodiodes and collection optics that have been confocallyaligned with the ablation light source is provided. FIG. 2 shows asensor system 60 for use in this embodiment. The system comprises alight source 64 for emitting a beam of light 68, which is directedthrough a delivery optics system 72 that focuses the beam at apreselected point 76, such as the surface of an individual's skin 80. Aportion of the light contacting the skin is reflected, and other lightis emitted from the irradiated area. A portion of this reflected andemitted light passes through a filter 84 and then through a collectionoptics system 88, which focuses the light on a phototodiode 92. Acontroller 96 is coupled to both the laser diode and the photodiode for,respectively, controlling the output of the laser diode and detectingthe light that reaches the photodiode. Only selected portions of thespectrum emitted from the skin pass through the filter. By analyzing theshifts in the reflected and emitted light from the targeted area, thesystem has the ability to detect when the stratum corneum has beenbreached, and this feedback is then used to control the light source,deactivating the pulses of light when the microporation of the stratumcorneum is achieved. By employing this type of active closed loopfeedback system, a self regulating, universally applicable device isobtained that produces uniformly dimensioned micropores in the stratumcorneum, with minimal power requirements, regardless of variations fromone individual to the next.

[0162] In another illustrative embodiment, a cooling device isincorporated into the system interface to the skin. FIG. 3A shows anillustrative schematic representation thereof. In this system 100, alight source 104 (coupled to a controller 106) emits a beam of light108, which passes through and is focused by a delivery optics system112. The beam is focused by the delivery optics system to a preselectedpoint 116, such as a selected area of an individual's skin 120. Acooling device 124, such as a Peltier device or other means of chilling,contacts the skin to cool the surface thereof. In a preferred embodimentof the cooling device 124 (FIG. 3B), there is a central hole 128 throughwhich the beam of focused light passes to contact the skin. Referringagain to FIG. 3A, a heat sink 132 is also preferably placed in contactwith the cooling device. By providing a cooling device with a small holein its center coincident with the focus of the light, the tissues in thegeneral area where the poration is to be created may be cooled to 5° C.to 10° C. This cooling allows a greater safety margin for the system tooperate in that the potential sensations to the user and the possibilityof any collateral damage to the epidermis directly below the porationsite are reduced significantly from non-cooled embodiment. Moreover, formonitoring applications, cooling minimizes evaporation of interstitialfluid and can also provide advantageous physical properties, such asdecreased surface tension of such interstitial fluid. Still further,cooling the tissue is known to cause a localized increase in blood flowin such cooled tissue, thus promoting diffusion of analytes from theblood into the interstitial fluid and promoting diffusion of deliveredpermeants away from the pore site or into the tissue underlying thepore.

[0163] The method can also be applied for other micro-surgery techniqueswherein the light-absorbing compound/heat-probe is applied to the areato be ablated and then the light source is used to selectively modulatethe temperature of the probe at the selected target site, affecting thetissues via the vaporization-ablation process produced.

[0164] A further feature of the invention is to use the light source tohelp seal the micropore after its usefulness has passed. Specifically,in the case of monitoring for an internal analyte, a micropore iscreated and some amount of interstitial fluid is extracted through thisopening. After a sufficient amount of interstitial fluid had beencollected, the light source is reactivated at a reduced power level tofacilitate rapid clotting or coagulation of the interstitial fluidwithin the micropore. By forcing the coagulation or clotting of thefluid in the pore, this opening in the body is effectively sealed, thusreducing the risk of infection. Also, the use of the light source itselffor both the formation of the micropore and the sealing thereof is aninherently sterile procedure, with no physical penetration into the bodyby any device or apparatus. Further, the thermal shock induced by thelight energy kills any microbes that may happen to be present at theablation site.

[0165] This concept of optical sterilization can be extended to includean additional step in the process wherein the light source is firstapplied in an unfocused manner, covering the target area with anilluminated area that extends 100 μm or more beyond the actual size ofthe micropore to be produced. By selecting the area over which theunfocused beam is to be applied, the flux density can be correspondinglyreduced to a level well below the ablation threshold but high enough toeffectively sterilize the surface of the skin. After a sufficiently longexposure of the larger area, either in one continuous step or in aseries of pulses, to the sterilizing beam, the system is then configuredinto the sharply focused ablation mode and the optical microporationprocess begins.

[0166] Another illustrative embodiment of the invention is to create therequired heat probe from a solid element, such as a small diameter wire.As in the previously described embodiment, the contacting surface of theheat probe must be able to have its temperature modulated from ambientbiological membrane temperatures to temperatures greater than 123° C.,within the required time allowed of, preferably, between about 1microsecond to 50 milliseconds at the high temperature (on-time) and atleast about 1 to 50 ms at the low temperature (off-time). In particular,being able to modulate the temperature up to greater than 150° C. for an“on” time of around 5 ms and an off time of 50 ms produces veryeffective thermal ablation with little or no sensation to theindividual.

[0167] Several methods for modulating the temperatures of the solidelement heat probe contact area may be successfully implemented. Forexample, a short length of wire may be brought up to the desired hightemperature by an external heating element such as an ohmic heatingelement used in the tip of a soldering iron. FIG. 4 shows an ohmicheating device 140 with a mechanical actuator. The ohmic heating devicecomprises an ohmic heat source 144 coupled to a solid element heat probe148. The ohmic heat source is also coupled through an insulating mount152 to a mechanical modulation device 156, such as a solenoid. In thisconfiguration, a steady state condition can be reached wherein the tipof the solid element probe will stabilize at some equilibriumtemperature defined by the physical parameters of the structure, i.e.,the temperature of the ohmic heat source, the length and diameter of thesolid element, the temperature of the air surrounding the solid element,and the material of which the solid element is comprised. Once thedesired temperature is achieved, the modulation of the temperature ofthe selected area of an organism's biological membrane 160 is effecteddirectly via the mechanical modulation device to alternatively place thehot tip of the wire in contact with the biological membrane for,preferably, a 5 ms on-time and then withdraw it into the air for,preferably, a 50 ms off-time.

[0168] Another illustrative example (FIG. 5), shows a device 170comprising a current source 174 coupled to a controller 178. The currentsource is coupled to a current loop 182 comprising a solid element 186formed into a structure such that it presents a high resistance point.Preferably, the solid element is held on a mount 190, and an insulator194 separates different parts of the current loop. The desiredmodulation of temperature is then achieved by merely modulating thecurrent through the solid element. If the thermal mass of the solidelement is appropriately sized and the heat sinking provided by theelectrodes connecting it to the current source is sufficient, thewarm-up and cool-down times of the solid element can be achieved in afew milliseconds. Contacting the solid element with a selected area ofbiological membrane 198 heats the biological membrane to achieve theselected ablation.

[0169] In FIG. 6 there is shown still another illustrative example ofporating the biological membrane with a solid element heat probe. Inthis system 200, the solid element 204 can be positioned within amodulatable alternating magnetic field formed by a coil of wire 208, theexcitation coil. By energizing the alternating current in the excitationcoil by means of a controller 212 coupled thereto, eddy currents can beinduced in the solid element heat probe of sufficient intensity that itwill be heated up directly via the internal ohmic losses. This isessentially a miniature version of an inductive heating system commonlyused for heat treating the tips of tools or inducing out-gassing fromthe electrodes in vacuum or flash tubes. The advantage of the inductiveheating method is that the energy delivered into the solid element heatprobe can be closely controlled and modulated easily via the electroniccontrol of the excitation coil with no direct electrical connection′ tothe heat probe itself. If the thermal mass of the solid element heatprobe and the thermal mass of the biological membrane in contact withthe tip of the probe are known, controlling the inductive energydelivered can allow precise control of the temperature at the contactpoint 216 with the biological membrane 220. Because the biologicalmembrane tissue is essentially non-magnetic at the lower frequencies atwhich inductive heating can be achieved, if appropriately selectedfrequencies are used in the excitation coil, then this alternatingelectromagnetic field will have no effect on the organism's tissues.

[0170] If a mechanically controlled contact modulation is employed, anadditional feature may be realized by incorporating a simple closed loopcontrol system wherein the electrical impedance between the probe tipand the subject's skin is monitored. In this manner, the probe can bebrought into contact with the subject's skin, indicated by the step-wisereduction in resistance once contact is made, and then held there forthe desired “on-time,” after which it can be withdrawn. Several types oflinear actuators are suitable for this form of closed loop control, suchas a voice-coil mechanism, a simple solenoid, a rotary system with a camor bell-crank, and the like. The advantage is that as the thermalablation progresses, the position of the thermal probe tip can besimilarly advanced into the biological membrane, always ensuring good acontact to facilitate the efficient transfer of the required thermalenergy. Also, for poration of skin, the change in the conductivityproperties of the stratum corneum and the epidermis can be used toprovide an elegant closed loop verification that the poration process iscomplete, i.e., when the resistance indicates that the epidermis hasbeen reached, it is time to stop the poration process. Similar changesin impedance can be used to control the depth of penetration to otherlayers as well.

[0171]FIG. 7 shows an illustrative example of such a closed loopimpedance monitor. In this system 230, there is an ohmic heat source 234coupled to a wire heat probe 238. The heat source is mounted through aninsulating mount 242 on a mechanical modulator 246. A controller 250 iscoupled to the wire and to the skin 254, wherein the controller detectschanges in impedance in the selected area 258 of skin, and when apredetermined level is obtained the controller stops the porationprocess.

[0172] Along the same line as hydraulic poration means are microlancetsadapted to just penetrate the stratum corneum for purposes ofadministering a permeant, such as a drug, through the pore formed or towithdraw an analyte through the pore for analysis. Such a device isconsidered to be “minimally invasive” as compared to devices and/ortechniques which are non-invasive. The use of micro-lancets thatpenetrate below the stratum corneum for withdrawing blood are wellknown. Such devices are commercially available from manufacturers suchas Becton-Dickinson and Lifescan and can be utilized in the presentinvention by controlling the depth of penetration. As an example of amicro-lancet device for collecting body fluids, reference is made toErickson et al., International Published PCT Application WO 95/10223(published Apr. 20, 1995). This application shows a device forpenetration into or through the dermal layer of the skin, withoutpenetration into subcutaneous tissues, to collect body fluids formonitoring, such as for blood glucose levels.

[0173] Poration of a biological membrane can also be accomplished usingsonic means. Sonic-poration is a variation of the optical meansdescribed above except that, instead of using a light source, a verytightly focused beam of sonic energy is delivered to the area of thestratum corneum to be ablated. The same levels of energy are required,i.e. a threshold of 70 mJ/cm²/50 ms still must be absorbed. The samepulsed focused ultrasonic transducers as described in parent applicationSer. Nos. 08/152,442 (now U.S. Pat. No. 5,458,140) and 08/152,174 (nowU.S. Pat. No. 5,445,611) can be utilized to deliver the required energydensities for ablation as are used in the delivery of sonic energy whichis modulated in intensity, phase, or frequency or a combination of theseparameters for the transdermal sampling of an analyte or the transdermaldelivery of drugs. This has the advantage of allowing use of the sametransducer to push a drug through the stratum corneum or pull a bodyfluid to the surface for analysis to be used to first create amicropore.

[0174] Additionally, electroporation or short bursts or pulses ofelectrical current can be delivered to the stratum corneum withsufficient energy to form micropores. Electroporation is known in theart for producing pores in biological membranes and electroporationinstruments are commercially available. Thus, a person of skill in thisart can select an instrument and conditions for use thereof withoutundue experimentation according to the guidelines provided herein.

[0175] The micropores produced in the biological membrane by the methodsof the present invention allow high flux rates of a variety of molecularweight therapeutic compounds to be delivered transmembranely. Inaddition, these non-traumatic microscopic openings into the body allowaccess to various analytes within the body, which can be assayed todetermine their internal concentrations.

EXAMPLE 1

[0176] In this example, skin samples were prepared as follows. Epidermalmembrane was separated from human cadaver whole skin by theheat-separation method of Klingman and Christopher, 88 Arch. Dermatol.702 (1963), involving the exposure of the full thickness skin to atemperature of 60 C for 60 seconds, after which time the stratum corneumand part of the epidermis (epidermal membrane) were gently peeled fromthe dermis.

EXAMPLE 2

[0177] Heat separated stratum corneum samples prepared according to theprocedure of Example 1 were cut into 1 cm² sections. These small sampleswere than attached to a glass cover slide by placing them on the slideand applying an pressure sensitive adhesive backed disk with a 6 mm holein the center over the skin sample. The samples were then ready forexperimental testing. In some instances the skin samples were hydratedby allowing them to soak for several hours in a neutral bufferedphosphate solution or pure water.

[0178] As a test of these untreated skin samples, the outputs of severaldifferent infrared laser diodes, emitting at roughly 810, 905, 1480 and1550 nanometers were applied to the sample. The delivery optics weredesigned to produce a focal waist 25 μm across with a final objectivehave a numerical aperture of 0.4. The total power delivered to the focalpoint was measured to be between 50 and 200 milliwatts for the 810 and1480 nm laser diodes, which were capable of operating in a continuouswave (CW) fashion. The 905 and 1550 nm laser diodes were designed toproduce high peak power pulses roughly 10 to 200 nanoseconds long atrepetition rates up to 5000 Hz. For the pulsed lasers the peak powerlevels were measured to be 45 watts at 905 nm and 3.5 watts at 1550 nm.

[0179] Under these operating conditions, there was no apparent effect onthe skin samples from any of the lasers. The targeted area wasilluminated continuously for 60 seconds and then examinedmicroscopically, revealing no visible effects. In addition, the samplewas placed in a modified Franz cell, typically used to test transdermaldelivery systems based on chemical permeation enhancers, and theconductivity from one side of the membrane to the other was measuredboth before and after the irradiation by the laser and showed no change.Based on these tests which were run on skin samples from four differentdonors, it was concluded that at these wavelengths the coupling of theoptical energy into or through the skin tissue was so small that noeffects are detectable.

EXAMPLE 3

[0180] To evaluate the potential sensation to a living subject whenilluminated with optical energy under the conditions of Example 2, sixvolunteers were used and the output of each laser source was applied totheir fingertips, forearms, and the backs of their hands. In the casesof the 810, 905 and 1550 nm lasers, the subject was unable to sense whenthe laser was turned on or off. In the case of the 1480 nm laser, therewas a some sensation during the illumination by the 1480 nm laseroperating at 70 mW CW, and a short while later a tiny blister was formedunder the skin due to the absorption of the 1480 nm radiation by one ofthe water absorption bands. Apparently the amount of energy absorbed wassufficient to induce the formation of the blister, but was not enough tocause the ablative removal of the stratum corneum. Also, the absorptionof the 1480 nm light occurred predominantly in the deeper, fullyhydrated (85% to 90% water content) tissues of the epidermis and dermis,not the relatively dry (10% to 15% water content) tissue of the stratumcorneum.

EXAMPLE 4

[0181] Having demonstrated the lack of effect on the skin in its naturalstate (Example 3), a series of chemical compounds was evaluated foreffectiveness in absorbing the light energy and then transferring thisabsorbed energy, via conduction, into or through the targeted tissue ofthe stratum corneum. Compounds tested included India ink; “SHARPIE”brand indelible black, blue, and red marking pens; methylene blue;fuschian red; epolite #67, an absorbing compound developed for moldinginto polycarbonate lenses for protected laser goggles; tincture ofiodine; iodine-polyvinylpyrrolidone complex (“BETADINE”); copperphthalocyanine; and printers ink.

[0182] Using both of the CW laser diodes described in Example 2,positive ablation results were observed on the in vitro samples ofheat-separated stratum corneum prepared according to Example 1 whenusing all of these products, however some performed better than others.In particular the copper phthalocyanine (CPC) and the epolite #67 weresome of the most effective. One probable reason for the superiorperformance of the CPC is its high boiling point of greater the 500° C.and the fact that it maintains its solid phase up to this temperature.

EXAMPLE 5

[0183] As copper phthalocyanine has already been approved by the FDA foruse in implantable sutures, and is listed in the Merck index as a ratherbenign and stabile molecule in regard to human biocompatability, thenext step taken was to combine the topical application of the CPC andthe focused light source to the skin of healthy human volunteers. Asuspension of finely ground CPC in isopropyl alcohol was prepared. Themethod of application used was to shake the solution and then apply asmall drop at the target site. As the alcohol evaporated, a fine anduniform coating of the solid phase CPC was then left on the surface ofthe skin.

[0184] The apparatus show in FIG. 1 was then applied to the site,wherein the CPC had been topically coated onto the skin, by placing theselected area of the individual's skin against a reference plate. Thereference plate consists of a thin glass window roughly 3 cm×3 cm, witha 4 mm hole in the center. The CPC covered area was then positioned suchthat it was within the central hole. A confocal video microscope(FIG. 1) was then used to bring the surface of the skin into sharpfocus. Positioning the skin to achieve the sharpest focus on the videosystem also positioned it such that the focal point of the laser systemwas coincident with the surface of the skin. The operator then activatedthe pulses of laser light while watching the effects at the target siteon the video monitor. The amount of penetration was estimated visuallyby the operator by gauging the amount of defocusing of the laser spot inthe micropore as the depth of the micropore increased, and this can bedynamically corrected by the operator, essentially following the ablatedsurface down into the tissues by moving the position of the camera/lasersource along the “z” axis, into the skin. At the point when the stratumcorneum had been removed down to the epidermis, the appearance of thebase of the hole changed noticeably, becoming much wetter and shinier.Upon seeing this change, the operator deactivated the laser. In manyinstances, depending on the state of hydration of the subject as well asother physiological conditions, a dramatic outflow of interstitial fluidoccurred in response to the barrier function of the stratum corneumbeing removed over this small area. The video system was used to recordthis visual record of the accessibility of interstitial fluid at theporation site.

EXAMPLE 6

[0185] The procedure of Example 5 was followed except that the CPC wasapplied to a transparent adhesive tape, which was then caused to adhereto a selected site on the skin of an individual. The results weresubstantially similar to those of Example 5.

EXAMPLE 7

[0186] Histology experiments were performed on cadaver skin according tomethods well known in the art to determine ablation threshold parametersfor given dye mixtures and collateral damage information. The topsurface of the skin sample was treated with a solution of copperphthalocyanine (CPC) in alcohol. After the alcohol evaporated, a topicallayer of solid phase CPC was distributed over the skin surface with amean thickness of 10 to 20 μm. FIG. 8A shows a cross-section of fullthickness skin prior to the laser application, wherein the CPC layer270, stratum corneum 274, and underlying epidermal layers 278 are shown.FIG. 8B shows the sample after a single pulse of 810 nm light wasapplied to an 80 um diameter circle with an energy density of 4000J/cm2, for a pulse period of 20 ms. It is noteworthy that there wasstill a significant amount of CPC present on the surface of the stratumcorneum even in the middle of the ablated crater 282. It should also benoted that laboratory measurements indicate that only about 10% of thelight energy incident on the CPC is actually absorbed, with the other90% being reflected or backscattered. Thus the effective energy fluxbeing delivered to the dye layer which could cause the desired heatingis only about 400 J/cm2. 8C shows the sample after 5 pulses of 810 nmlight were applied, wherein the stratum corneum barrier was removed withno damage to the underlying tissue. These results are a goodrepresentation of the “ideal” optically modulated thermal ablationperformance. FIG. 8D shows the sample after 50 pulses were applied.Damaged tissue 286 was present in the epidermal layers due tocarbonization of non ablated tissue and thermal denaturing of theunderlying tissue. FIGS. 8A-8C show separations between the stratumcorneum and the underlying epidermal layers due to an artifact ofdehydration, freezing, and preparations for imaging.

EXAMPLE 8

[0187] To examine the details of the thermal ablation mechanism, amathematical model of the skin tissues was constructed upon whichvarious different embodiments of the thermal ablation method could betried. This model computes the temperature distribution in a layeredsemi-infinite medium with a specified heat flux input locally on thesurface and heat removal from the surface some distance away, i.e.convection is applied between the two. The axisymmetric, time-dependentdiffusion equation is solved in cylindrical coordinates using thealternating-direction-implicit (ADI) method. (Note: Constant Temp. B.C.is applied on lower boundary to serve as z->inf; and zero radial heatflux is applied on max radial boundary to serve as r->inf). The layersare parallel to the surface and are defined as: (1) dye; (2) stratumcorneum; (3) underlying epidermis; and (4) dermis. The depth into thesemi-infinite medium and thermal properties, density (rho), specificheat (c), and conductivity (k) must be specified for each layer.

[0188] First, a heat-transfer coefficient, h, on the skin is computedbased on the “steady,” “1-D,” temperature distribution determined by theambient air temperature, skin surface temperature, and dermistemperature. It is assumed that there is no dye present and provides “h”on the skin surface. The program then allows one to use this “h” on thedye layer surface or input another desired “h” for the dye surface.Next, the “steady” temperature distribution is computed throughout alllayers (including the dye layer) using the specified “h” at the dyesurface. This temperature distribution is the initial condition for thetime-dependent heating problem. This constitutes the “m-file” initial.m.The program then solves for the time-dependent temperature distributionby marching in time, computing and displaying the temperature field ateach step.

[0189] Each embodiment of the method described herein, for whichempirical data have been collected, has been modeled for at least oneset of operational parameters, showing how stratum corneum ablation canbe achieved in a precise and controllable fashion. The output of thesimulations is presented graphically in two different formats: (1) across-sectional view of the skin showing the different tissue layerswith three isotherms plotted on top of this view which define threecritical temperature thresholds, and (2) two differenttemperature-vs-time plots, one for the point in the middle of thestratum corneum directly beneath the target site, and the second for thepoint at the boundary of the viable cell layers of the epidermis and theunderside of the stratum corneum. These plots show how the temperatureat each point varies with time as the heat pulses are applied as if onecould implant a microscopic thermocouple into the tissues. In addition,the application of this model allows investigation of the parametriclimits within which the method can be employed to set the outer limitsfor two important aspects of the methods performance. First, generalcases are presented cases that define the envelope within which themethod can be employed without causing pain or undesired tissue damage.

[0190] For any given heat source, as described in the several differentembodiments of the invention, there is a point at which the effect onthe subject's skin tissues becomes non-optimal in that the subjectperceives a pain sensation, or that the viable cells in the underlyingepidermis and/or dermis sustain temperatures, which if maintained for along enough duration, will render damage to these tissues. Accordingly,a test simulation was run using the optically heated topical copperphthalocyanine (CPC) dye embodiment as a baseline method to establishhow the thermal time constants of the different skin tissue layersessentially define a window within which the method can be employedwithout pain or damage to adjacent tissue layers.

[0191]FIGS. 9 and 10 show schematic cross-sectional views of the skinand the topical dye layer. In each figure, three distinct isotherms aredisplayed: (1) 123 C, the point at which vaporization of the water inthe tissue produces an ablation of the tissue; (2) 70 C, the point atwhich viable cells will be damaged if this temperature is maintained forseveral seconds; and (3) 45 C, the average point at which a sensation ofpain will be perceived by the subject. This pain threshold is describedin several basic physiology texts, but experience shows this thresholdto be somewhat subjective. In fact, in repeated tests on the sameindividual, different poration sites within a few millimeters of eachother can show significantly different amounts of sensation, possiblydue to the proximity to a nerve ending in relationship to the porationsite.

[0192] The dimensions on the graphs show the different layers of the dyeand skin, as measured in m, with flat boundaries defining them. Whereasthe actual skin tissues have much more convoluted boundaries, in a meansense for the dimensions involved, the model provides a goodapproximation of the thermal gradients present in the actual tissues.The dimensions used in this, and all subsequent simulations, for thethicknesses of the CPC dye layer and the various skin layers are asfollows: dye, 10 m; stratum corneum, 30 m; underlying epidermis, 70 m;and dermis, 100 m.

[0193] Additional conditions imposed on the model for this particularsimulation are shown in the following tables: TABLE 1 Initial Conditionsfor Finite Difference Thermal Model Ambient Air Temperature Ta = 20 C.Skin Surface Temperature Ts = 30 C. Dermis Temperature Td = 37 C. DyeVaporization Temperature Tvap = 550 C. S.C. Vaporization Temperature Tc1= 123 C. Tissue Damage Temperature Tc2 = 70 C. “Pain” Temperature Tc3 =45 C. Radius of Irradiated Area R_(hot) = 30 m Energy Density AppliedFLUX = 400 Joules/cm²

[0194] TABLE 2 Parameter Dye S.C. Epidermis Dermis Thermal 0.00046.00123 0.00421 0.00421 Conductivity Density 0.67 1.28 1.09 1.09 SpecificHeat 0.8 1.88 3.35 3.35

[0195] When these simulations are run, the following conservativeassumptions are imposed:

[0196] 1. While some portion of the stratum corneum may be shown ashaving a temperature already exceeded the ablation threshold for thermalvaporization of the water content, this event is not modeled, and thesubsequent loss of heat energy in the tissues due to this vaporizationis not factored into the simulation. This will cause a slight elevationin the temperatures shown in the underlying tissues from that point onin the simulation run.

[0197] 2. Similarly, when some portion of the copper phthalocyanine(CPC) dye layer is shown to have reached its vaporization point of 550°C., this event is not modeled, but the temperature is merelyhard-limited to this level. This will also cause a slight elevation ofthe subsequent temperatures in the underlying layers as the simulationprogresses.

[0198] Even with these simplifications used in the model, thecorrelation between the predicted performance and the empiricallyobserved performance based on both clinical studies and histologicalstudies on donor tissue samples is remarkable. The key data to note inFIGS. 9 and 10 are the length of time which the heat pulse is applied,and the location of the three different threshold temperatures displayedby the isotherms.

[0199] In FIG. 9, with a pulse length of 21 milliseconds, the 70° C.isotherm just crosses the boundary separating the stratum corneum andthe viable cell layers in the epidermis. In in vitro studies on donorskin samples under these conditions, fifty pulses of thermal energydelivered 50 milliseconds apart cause detectable damage to this toplayer of living cells (see FIG. 8D). However, it was also shown in thein vitro studies that five pulses of heat energy at these same operatingparameters, did not produce any significant damage to these tissues. Itseems reasonable that even though the nominal damage threshold may havebeen exceeded, at least in a transient sense, this temperature must bemaintained for some cumulative period of time to actually cause anydamage to the cells. Nevertheless, the basic information presented bythe simulation is that if one keeps the “on-time” of the heat pulse toless than 20 milliseconds with the flux density of 400 Joules/cm², thenno damage to the living cells in the underlying epidermis will besustained, even though the ablation threshold isotherm has been movedwell into or through the stratum corneum. In other words, by using a lowflux density thermal energy source, modulated such that the “on time” issuitably short, ablation of the stratum corneum can be achieved withoutany damage to the adjacent cells in the underlying epidermis (see FIG.8C). This is possible in large part due to the significantly differentthermal diffusivities of these two tissues layers. That is, the stratumcorneum, containing only about 10% to 20% water content, has a muchlower thermal conductivity constant, 0.00123 J/(S*cm*K), than the0.00421J/(S*cm*K) of the epidermis. This allows the temperature to buildup in the stratum corneum, while maintaining a tight spatial definition,to the point at which ablation will occur.

[0200] In FIG. 10, the same simulation scenario started in the damagethreshold critical point run illustrated in FIG. 9 is carried outfarther in time. By leaving the heat pulse on for 58 milliseconds at thesame flux density of 400 Joules/cm² within the 60 μm diameter circle ofdye being heated, the pain sensory isotherm at 45° C. just enters theinnervated layer of skin comprised by the dermis. In addition, thedamage threshold isotherm moves significantly farther into the epidermallayer than where it was shown to be in FIG. 9. Relating this simulationto the numerous clinical studies conducted with this method, anexcellent verification of the model's accuracy is obtained in that themodel shows almost exactly the duration of ‘on-time’ that the heat probecan be applied to the skin before the individual feels it. In clinicaltests, a controllable pulse generator was used to set the “on-time” and“off-time” of a series of light pulses applied to the topical layer ofcopper phthalocyanine (CPC) dye on the skin. While maintaining aconstant “off-time” of 80 milliseconds, the “on-time” was graduallyincreased until the subject reported a mild “pain” sensation. Withoutexception, all of the subjects involved in these studies, reported thefirst “pain” at an “on-time” of between 45 and 60 milliseconds, veryclose to that predicted by the model. In addition, the site-to-sitevariability mentioned previously as regards the sensation of “pain” wasnoted in these clinical studies. Accordingly, what is reported as “pain”is the point at which the first unambiguous sensation is noticeable. Atone site this may be reported as pain, whereas at an adjacent site thesame subject may report this as merely “noticeable.”

[0201] One element of this clinical research is the realization thateven at the same site, a non-uniform pulse-train of heat pulses may workwith the subject's psycho-physiological neuro-perception to cause agenuine reduction in perceived sensation. For example, a series ofshorter length heat pulses can be used to saturate the neurons in thearea, momentarily depleting the neuro-transmitters available at thissynaptic junction and therefore limiting the ability to send a “pain”message. This then allows a longer pulse following these short pulses tobe less noticeable than if it were applied at the beginning of thesequence. Accordingly, a series of experiments was conducted with somearbitrarily created pulse trains, and the results were consistent withthis hypothesis. An analogy for this situation might be found in theperception when one first steps into a very hot bath that is painful atfirst, but quickly becomes tolerable as one acclimates to the heatsensation.

EXAMPLE 9

[0202] An object of this invention is to achieve a painless,micro-poration of the stratum corneum without causing any significantdamage to the adjacent viable tissues. As described in the simulationillustrated in Example 8 and FIGS. 9-10, a boundary appears to exist forany given flux density of thermal energy within the ablation target spotwithin which the micro-poration can be achieved in just such a painlessand non-traumatic manner. Both the in vivo and in vitro studies haveshown that this is the case, and this has permitted development throughempirical methods of some operational parameters that appear to workvery well. The following set of simulations shows how the method workswhen these specific parameters are used.

[0203] In the first case, a pulse train of ten pulses, 10 milliseconds“on-time” separated by 10 milliseconds “off-time” is applied to theCPC-covered skin. FIG. 11 shows the final temperature distribution inthe skin tissues immediately after this pulse train has ended. As can beseen, the isotherms representing the three critical temperaturethresholds show that stratum corneum ablation has been achieved, with nosensation present in the dermal layer nerves and very little cross-overof the damage threshold into or through the viable cells of theunderlying epidermis. As mentioned previously, it appears that toactually do permanent cell damage, the epidermal cells must not only beheated up to a certain point, but they also must be held at thistemperature for some period of time, generally thought to be about fiveseconds. FIGS. 12 and 13 show the temperature of the stratum corneum andthe viable epidermis, respectively, as a function of time, showingheating during the “on-time” and cooling during the “off-time” for theentire ten cycles. Relating this simulation to the in vivo studiesconducted, note that in better than 90% of the poration attempts withthe system parameters set to match the simulation, effective poration ofthe stratum corneum was achieved without pain to the subject, and insubsequent microscopic examination of the poration site several dayslater, no noticeable damage to the tissues was apparent. The in vitrostudies conducted on whole thickness donor skin samples were alsoconsistent with the model's prediction of behavior.

EXAMPLE 10

[0204] In conducting both the empirical in vivo studies, and thesesimulations, it appears that prechilling of the skin aids in optimizingthe micro-poration process for reducing the probability of pain ordamage to adjacent tissues. In practice, this can easily be achievedusing a simple cold-plate placed against the skin prior to the porationprocess. For example, applying a Peltier cooled plate to the 1 cmdiameter circle surrounding the poration target site, with the plateheld at roughly 5° C. for a few seconds, significantly reduces thetemperature of the tissues. A schematic illustration of an experimentaldevice used for this purpose in the laboratory is shown in FIGS. 3A-B.By applying exactly the same ten-cycle pulse train as used in the runillustrated in Example 9, one can see, by comparing FIG. 11 to FIG. 14,FIG. 12 to FIG. 15, and FIG. 13 to FIG. 16, how much improvement can bemade in the control of the temperature penetration into or through theskin tissues. Once again, the relatively low thermal diffusivity andspecific heat of the stratum corneum as compared to the epidermis anddermis is advantageous. Once cooled, the highly hydrated tissues of theepidermis and dermis require a much larger thermal energy input toelevate their temperatures, whereas the stratum corneum, with itsrelatively dry makeup, can quickly be heated up to the ablationthreshold.

EXAMPLE 11

[0205] Once the basic thermal conduction mechanism of delivering theenergy into or through the skin tissues underlying the effectivepainless ablation and micro-poration of the stratum corneum isunderstood, several different specific methods to achieve the requiredrapid temperature modulations of the contact point can be conceived,such as the hot wire embodiments illustrated in FIGS. 4-7.

[0206] A basic embodiment, as described herein, uses an Ohmic heatingelement (FIG. 4), such as the tip of a small cordless soldering iron,with a suitably sized, relatively non-reactive, wire wrapped around itwith a short amount of the wire left to protrude away from the body ofthe heater. When electricity is applied with a constant current source,the heater will come up to some temperature and within a few seconds,achieve a steady state with the convection losses to the surroundingair. Similarly, the wire, which is a part of this thermal system, willreach a steady state such that the very tip of the wire can be raised toalmost any arbitrary temperature, up to roughly 1000° C. with thesetypes of components. The tip can be sized to give exactly the dimensionmicropore desired.

[0207] In the laboratory, tungsten wires with a diameter of 80 μmattached to the replaceable tip of a “WAHL” cordless soldering iron withapproximately 2 mm of wire protruding from the tip have been utilized.With a thermocouple, the temperature of the tip has been measured at itssteady state, and it has been noted that by varying the constant currentsettings, steady state temperatures of greater than 700° C. can easilybe reached. To achieve the desired modulation, a low mass, fast responseelectromechanical actuator was coupled to the tip such that the positionof the wire could be translated linearly more than 2 mm at up to a 200Hz rate. Then, by mounting the entire apparatus on a precision stage,this vibrating tip could very controllably be brought into contact withthe skin surface in a manner where it was only in contact for less than10 milliseconds at a time, the “on-time,” while an “off-time” ofarbitrarily long periods could be achieved by setting the pulsegenerator accordingly. These in vivo studies showed that the porationcould actually be achieved before the subject being porated even knewthat the tip of the wire was being brought into contact with the skin.

[0208] To compare the performance of this embodiment to the opticallyheated topical CPC dye embodiment, the following simulations were runaccording to the procedure of Example 8. Essentially, by only varyingthe initial conditions, the hot wire embodiment can be run with theidentical simulation code. Because the contact with the wire occursessentially instantly, there is no time dependent build-up of heat inthe CPC dye layer and when the wire is physically removed from contactwith the skin, there is a no residual heat still left on the surface asthere is with the heated CPC dye layer. Also, as the wire itself definesthe area targeted for ablation/micro-poration, there should be nolateral diffusion of thermal energy prior to its application to thestratum corneum. The comparative performances of the “hot-wire”embodiment are shown in FIGS. 17-19.

EXAMPLE 12

[0209] In this example, the procedure of Example 11 was followed exceptthat the skin was pre-cooled according to the procedure of Example 10.Similarly, pre-cooling the target site yields similarly positive resultswith the “hot-wire” embodiment. The results of the pre-cooled simulationof the “hot-wire” approach are shown in FIGS. 20-22.

EXAMPLE 13

[0210] As discussed in the background introduction of this disclosure,the Tankovich '803 patent appears at first glance to be similar to thepresently claimed invention. In this example, the simulation model wasset up with the operating parameters specified in Tankovich '803, i.e. apulse width of 1 s and a power level of 40,000,000 W/cm². FIGS. 23 and24 show that under these conditions no portion of the stratum corneumreaches the threshold for flash vaporization of water, 123 C, and thusno ablation/microporation of the stratum corneum occurs. In practice,applying this type of high peak power, short duration pulse to thetopical dye layer merely vaporizes the dye off of the surface of theskin with no effect on the skin. This example, thus, demonstrates thatthe conditions specified by Tankovich '803 are inoperative in thepresently claimed invention.

EXAMPLE 14

[0211] In this example, interstitial fluid obtained after porating theskin according to the procedure of Example 6 was collected and analyzedto determine the glucose concentration thereof. Data were obtained onfour non-diabetic subjects and six type I diabetic subjects undergoing aglucose load test. Subject's ages ranged from 27 to 43. The goal of thestudy was to examine the utility of the method for painlessly harvestingenough interstitial fluid (ISF) from the subjects to allow the ISFsamples to be assayed for glucose content, and then compare theseconcentrations to the glucose level presenting in the subject's wholeblood.

[0212] All subjects had both the blood and ISF glucose assays performedwith the “ELITE” system from Miles-Bayer. All ten subjects underwentidentical measurement protocols, with adjustments being made regardingthe glucose load and insulin shot for those subjects with insulindependent diabetes.

[0213] The basic design of the study was to recruit a modest number ofvolunteers, some with diabetes and some without diabetes, from which aseries of sample pairs of ISF and whole blood were drawn every 3 to 5minutes throughout the 3 to 4 hour duration of the study period. Boththe blood and the ISF samples were assayed for glucose and thestatistical relationship between the blood glucose levels and theinterstitial fluid determined. To examine the hypothesized temporal lagof the ISF glucose levels as compared to the whole blood glucose levels,the study subjects were induced to exhibit a significant and dynamicchange in their glucose levels. This was accomplished by having eachsubject fast for 12 hours prior to beginning the test and then givingthe subject a glucose load after his or her baseline glucose levels havebeen established via a set of three fasting blood and ISF glucoselevels. After the baseline levels had been established, the subjectswere given a glucose load in the form of sweet juice based on thefollowing guidelines:

[0214] i. For the control subjects, the glucose load was calculatedbased on a 0.75 gram glucose per pound of body weight.

[0215] ii. For the subjects with insulin dependent diabetes the glucoseload was 50 grams of glucose. In addition, immediately after taking theglucose load the diabetic subjects will self inject their normal morningdose of fast acting insulin. In the case where the diabetic subjectpresents with fasting glucose levels above 300 mg/dL, they were asked togive themselves their insulin injection first, and the glucose load wasprovided after their blood glucose levels have dropped to below 120mg/dL.

[0216] Each subject recruited was first given a complete description ofthe study in the “Informed Consent” document which they were required tounderstand and sign before they were officially enrolled into theprogram. Upon acceptance, they completed a medical historyquestionnaire. The detailed clinical procedure implemented was:

[0217] (a) Subject fasted from 9:00 p.m. the night before the studyvisit, consuming only water. No caffeine, cigarettes, fruit juice wereallowed during this period.

[0218] (b) Subject arrived at the testing facility by 9:00 a.m. the nextday.

[0219] (c) Subject was seated in a reclining chair provided for thesubject to relax in throughout the study procedure.

[0220] (d) Both whole blood and ISF samples were taken at three to fiveminute intervals beginning upon the subject's arrival and continuing forthe next three to four hours. The duration over which the data werecollected was based on when the subject's blood glucose levels hadreturned to the normal range and stabilized after the glucose load. TheISF samples were harvested using the optical poration, ISF pumpingmethod, described in more detail below. Each ISF sample was roughly 5 PLby volume to ensure a good fill of the ELITE test strip. The bloodsamples were obtained via a conventional finger prick lancet. Both theISF and the blood samples were immediately assayed for glucose with theELITE home glucometer system from Miles-Bayer. To improve the estimateof the ‘true’ blood glucose levels, two separate ELITE assays were bedone on each finger stick sample.

[0221] (e) To facilitate the continued collection of the ISF from thesame site throughout the entire data collection phase for a givenindividual, a 5 by 5 matrix of twenty five micropores was created on thesubject's upper forearm, each micropore being between 50 and 80 μmacross and spaced 300 μm apart. A 30 μm diameter teflon disk with a 6 mmhole in the center was attached to the subject's forearm with a pressuresensitive adhesive and positioned such that the 6 mm center hole waslocated over the 5 by 5 matrix of micropores. This attachment allowed aconvenient method by which a small suction hose could be connected,applying a mild vacuum (10 to 12 inches of Hg) to the porated area toinduce the ISF to flow out of the body through the micropores. The topof the teflon disk was fitted with a clear glass window allowing theoperator to directly view the micro-porated skin beneath it. When a 5 pLbead of ISF was formed on the surface of the skin, it could easily beascertained by visually monitoring the site through this window. Thislevel of vacuum created a nominal pressure gradient of around 5pounds/square inch (PSI). Without the micropores, no ISF whatsoevercould be drawn from the subject's body using only the mild vacuum.

[0222] (f) After the first three sample pairs have been drawn, thesubject was given a glucose load in the form of highly sweetened orangejuice. The amount of glucose given was 0.75 grams per pound of bodyweight for the nondiabetic subjects and 50 grams for the diabeticsubjects. The diabetic subjects also self administered a shot of fastacting insulin, (regular) with the dosage appropriately calculated,based on this 50 gram level of glucose concurrent with the ingestion ofthe glucose load. With the normal 1.5 to 2.5 hour lag between receivingan insulin shot and the maximum effect of the shot, the diabeticsubjects were expected to exhibit an upwards excursion of their bloodglucose levels ranging up to 300 mg/dL and then dropping rapidly backinto the normal range as the insulin takes effect. The nondiabeticsubjects were expected to exhibit the standard glucose tolerance testprofiles, typically showing a peak in blood glucose levels between 150mg/dL and 220 mg/dL from 45 minutes to 90 minutes after administeringthe glucose load, and then a rapid drop back to their normal baselinelevels over the next hour or so.

[0223] (g) Following the administration of the glucose load or glucoseload and insulin shot, the subjects had samples drawn, simultaneously,of ISF and finger prick whole blood at five minute intervals for thenext three to four hours. The sampling was terminated when the bloodglucose levels in three successive samples indicate that the subject'sglucose had stabilized.

[0224] Upon examination of the data, several features were apparent. Inparticular, for any specific batch of ELITE test strips, there exist adistinct shift in the output shown on the glucometer in mg/dL glucose ascompared to the level indicated on the blood. An elevated reading wouldbe expected due to the lack of hematocrit in the ISF and to the normaldifferences in the electrolyte concentrations between the ISF and wholeblood. Regardless of the underlying reasons for this shift in output, itwas determined via comparison to a reference assay that the true ISFglucose levels are linearly related to the values produced by the ELITEsystem, with the scaling coefficients constant for any specific batch ofELITE strips. Consequently, for the comparison of the ISF glucose levelsversus the whole blood measurements, first order linear correction wasapplied to the ISF data as follows:

ISF _(glucose)=0.606*ISF _(ELITE)+19.5.

[0225] This scaling of the output of the ELITE glucometer when used tomeasure ISF glucose levels, allows one to examine, over the entire dataset, the error terms associated with using ISF to estimate blood glucoselevels. Of course, even with no linear scaling whatsoever, thecorrelations between the ISF glucose values and the blood glucose levelsare the same as the scaled version.

[0226] Based on the majority of the published body of literature on thesubject of ISF glucose as well as preliminary data, it was originallyexpected that a 15 to 20 minute lag between the ISF glucose levels andthe those presented in the whole blood from a finger stick would beobserved. This is not what the data showed when analyzed. Specifically,when each individual's data set is analyzed to determine the time shiftrequired to achieve the maximum correlation between the ISF glucoselevels and the blood glucose levels it was discovered that the worstcase time lag for this set of subjects was only 13 minutes and theaverage time lag was only 6.2 minutes, with several subjects showing atemporal tracking that was almost instantaneous (about 1 minute).

[0227] Based on the minimal amount of lag observed in this data set, thegraph shown in FIG. 25 presents all ten of the glucose load tests,concatenated one after another on an extended time scale. The data arepresented with no time shifting whatsoever, showing the high level oftracking between the ISF and blood glucose levels the entire clinicaldata set being dealt with in exactly the same manner. If the entire dataset is shifted as a whole to find the best temporal tracking estimate,the correlation between the ISF and blood glucose levels peaks with adelay of two (2) minutes at an r value of r=0.97. This is only a trivialimprovement from the unshifted correlation of r=0.964. Therefore, forthe remainder of the analysis the ISF values are treated with no timeshift imposed on them. That is, each set of blood and ISF glucose levelsis dealt with as simultaneously collected data pairs.

[0228] After the unshifted Elite ISF readings had been scaled to reflectthe proportional glucose present in the ISF, it was possible to examinethe error associated with these data. The simplest method for this is toassume that the average of the two ELITE finger-stick blood glucosereadings is in fact the absolutely correct value, and then to merelycompare the scaled ISF values to these mean blood glucose values. Thesedata are as follows: Standard Deviation Blood-ISF, 13.4 mg/dL;Coefficient of Variance of ISF, 9.7%; Standard Deviation of the TwoElites, 8.3 mg/dL; and Coefficient of Variance of Blood (Miles), 6%.

[0229] As these data show, the blood based measurement already containsan error term. Indeed, the manufacturer's published performance dataindicates that the ELITE system has a nominal Coefficient of Variance(CV) of between 5% and 7%, depending on the glucose levels and theamount of hematocrit in the blood.

[0230] An additional look at the difference term between the ISF glucoseand the blood glucose is shown in the form of a scatter plot in FIG. 26.In this figure, the upper and lower bounds of the 90% confidenceinterval are also displayed for reference. It is interesting to notethat with only two exceptions, all of the data in the range of bloodglucose levels below 100 mg/dL fall within these 90% confidence intervalerror bars. This is important as the consequences of missing a trendtowards hypoglycemia would be very significant to the diabetic user.That is, it would be much better to under-predict glucose levels in the40 to 120 mg/dL than to over predict them.

[0231] Essentially, if one assumes that the basic assay error when theELITE system is used on ISF is comparable to the assay error associatedwith the ELITE's use on whole blood, then the Deviation of the ISFglucose from the blood glucose can be described as:

ISF _(deviation)=[(ISF _(actual))+(ISF _(actual))²]^(1/2).

[0232] Applying this equation to the values shown above, one can solvefor the estimated ‘true’ value of the ISF error term:

ISF _(actual)=[(ISF _(deviation))²−(Blood_(actual))²]^(1/2).

[0233] Or, solving the equation,

ISF _(actual)=[(13.4)²−(8.3)²]^(1/2)=10.5 mg/dl.

[0234] A histogram of the relative deviation of the ISF to the bloodglucose levels is shown in FIG. 27.

[0235] Drug Delivery through Pores in the Biological Membrane

[0236] The present invention also includes a method for the delivery ofdrugs, including drugs currently delivered transmembrane, throughmicropores in the stratum corneum or other biological membrane. In oneillustrative embodiment, the delivery is achieved by placing thesolution in a reservoir over the poration site. In another illustrativeembodiment, a pressure gradient is used to further enhance the delivery.In still another illustrative embodiment, sonic energy is used with orwithout a pressure gradient to further enhance the delivery. The sonicenergy can be operated according to traditional transdermal parametersor by utilizing acoustic streaming effects, which will be describedmomentarily, to push the delivery solution through the poratedbiological membrane.

EXAMPLE 15

[0237] This example shows the use of stratum corneum poration for thedelivery of lidocaine, a topical analgesic. The lidocaine solution alsocontained a chemical permeation enhancer formulation designed to enhanceits passive diffusion across the stratum corneum. A drawing of anillustrative delivery apparatus 300 is shown in FIG. 28, wherein theapparatus comprises a housing 304 enclosing a reservoir 308 for holdinga drug-containing solution 312. The top portion of the housing comprisesan ultrasonic transducer 316 for providing sonic energy to aid intransporting the drug-containing solution through micropores 320 in thestratum corneum 324. A port 328 in the ultrasonic transducer permitsapplication of pressure thereto for further aiding in transporting thedrug-containing solution through the micropores in the stratum corneum.The delivery apparatus is applied to a selected area of an individual'sskin such that it is positioned over at least one, and preferably aplurality, of micropores. An adhesive layer 332 attached to a lowerportion of the housing permits the apparatus to adhere to the skin suchthat the drug-containing solution in the reservoir is in liquidcommunication with the micropores. Delivery of the drug through themicropores results in transport into the underlying epidermis 336 anddermis 340.

[0238] Five subjects were tested for the effectiveness of drug deliveryusing poration together with ultrasound. The experiment used two siteson the subjects left forearm about three inches apart, equally spacedbetween the thumb and upper arm. The site near the thumb will bereferred to as site 1 the site furthest from the thumb will be referredto as site 2. Site 1 was used as a control where the lidocaine andenhancer solution was applied using an identical delivery apparatus 300,but without any micro-poration of the stratum corneum or sonic energy.Site 2 was porated with 24 holes spaced 0.8 millimeters apart in a gridcontained within a 1 cm diameter circle. The micropores in Site 2 weregenerated according to the procedure of Example 6. Lidocaine and lowlevel ultrasound were applied. Ultrasound applications were made with acustom manufactured Zevex ultrasonic transducer assembly set in burstmode with 0.4 Volts peak to peak input with 1000 count bursts occurringat 10 Hz with a 65.4 kHz fundamental frequency, i.e., a pulse modulatedsignal with the transducer energized for 15 millisecond bursts, and thenturned off for the next 85 milliseconds. The measured output of theamplifier to the transducer was 0.090 watts RMS.

[0239] After application of the lidocaine, sensation measurements weremade by rubbing a 30 gauge wire across the test site. Experiments wereexecuted on both sites, Site 1 for 10 to 12 minute duration and Site 2for two 5 minute duration intervals applied serially to the same site.Both sites were assessed for numbness using a scale of 10 to 0, where 10indicated no numbness and 0 indicated complete numbness as reported bythe test subjects. The following summary of results is for all 5subjects.

[0240] The control site, site 1, presented little to no numbness (scale7 to 10) at 10 to 12 minutes. At approximately 20 minutes some numbness(scale 3) was observed at site 1 as the solution completely permeatedthe stratum corneum. Site 1 was cleaned at the completion of thelidocaine application. Site 2 presented nearly complete numbness (scale0 to 1) in the 1 cm circle containing the porations. Outside the 1 cmdiameter circle the numbness fell off almost linearly to 1 at a 2.5 cmdiameter circle with no numbness outside the 2.5 cm diameter circle.Assessment of site 2 after the second application resulted in a slightlylarger totally numb circle of about 1.2 cm diameter with numbnessfalling off linearly to 1 in an irregular oval pattern with a diameterof 2 to 2.5 cm perpendicular to the forearm and a diameter of 2 to 6 cmparallel to the forearm. Outside the area no numbness was noted. Agraphic representation of illustrative results obtained on a typicalsubject is shown in FIGS. 29A-C. FIGS. 29A and 29B show the resultsobtained at Site 2 (porated) after 5 and 10 minutes, respectively. FIG.29C shows the results obtained at Site 1 (control with no poration).

[0241] Sonic Energy and Enhancers for Enhancing Transdermal Flux

[0242] The physics of sonic energy fields created by sonic transducerscan be utilized in a method by which sonic frequency can be modulated toimprove on flux rates achieved by other methods. As shown in FIG. 1 ofU.S. Pat. No. 5,445,611, hereby incorporated herein by reference, theenergy distribution of an sonic transducer can be divided into near andfar fields. The near field, characterized by length N, is the zone fromthe first energy minimum to the last energy maximum. The zone distal tothe last maximum is the far field. The near (N) field pattern isdominated by a large number of closely spaced local pressure peaks andnulls. The length of the near field zone, N, is a function of thefrequency, size, and shape of the transducer face, and the speed ofsound in the medium through which the ultrasound travels. For a singletransducer, intensity variations within its normal operating range donot affect the nature of the sonic energy distribution other than in alinear fashion. However, for a system with multiple transducers, allbeing modulated in both frequency and amplitude, the relativeintensities of separate transducers do affect the energy distribution inthe sonic medium, regardless of whether it is skin or another medium.

[0243] By changing the frequency of the sonic energy by a modest amount,for example in the range of about 1 to 20%, the pattern of peaks andnulls remains relatively constant, but the length N of the near fieldzone changes in direct proportion to the frequency. Major changes thefrequency, say a factor of 2 or more, will most likely produce adifferent set of resonances or vibrational modes in the transducer,causing a significantly and unpredictably different near field energypattern. Thus, with a modest change in the sonic frequency, the complexpattern of peaks and nulls is compressed or expanded in anaccordion-like manner. By selecting the direction of frequencymodulation, the direction of shift of these local pressure peaks, can becontrolled. By applying sonic energy at the surface of the skin,selective modulation of the sonic frequency controls movement of theselocal pressure peaks through the skin either toward the interior of thebody or toward the surface of the body. A frequency modulation from highto low drives the pressure peaks into the body, whereas a frequencymodulation from low to high pulls the pressure peaks from within thebody toward the surface and through the skin to the outside of the body.

[0244] Assuming typical parameters for this application of, for example,a 1.27 cm diameter sonic transducer and a nominal operating frequency of10 MHz and an acoustic impedance similar to that of water, a frequencymodulation of 1 MHz produces a movement of about 2.5 mm of the peaks andnulls of the near field energy pattern in the vicinity of the stratumcorneum. From the perspective of transdermal and/or transmucosalwithdrawal of analytes, this degree of action provides access to thearea well below the stratum corneum and even the epidermis, dermis, andother tissues beneath it. For any given transducer, there may be anoptimal range of frequencies within which this frequency modulation ismost effective.

[0245] The flux of a drug or analyte across the skin can also beincreased by changing either the resistance (the diffusion coefficient)or the driving force (the gradient for diffusion). Flux can be enhancedby the use of so-called penetration or chemical enhancers.

[0246] Chemical enhancers are comprised of two primary categories ofcomponents, i.e., cell-envelope disordering compounds and solvents orbinary systems containing both cell-envelope disordering compounds andsolvents.

[0247] Cell envelope disordering compounds are known in the art as beinguseful in topical pharmaceutical preparations and function also inanalyte withdrawal through the skin. These compounds are thought toassist in skin penetration by disordering the lipid structure of thestratum corneum cell-envelopes. A comprehensive list of these compoundsis described in European Patent Application 43,738, published Jun. 13,1982, which is incorporated herein by reference. It is believed that anycell envelope disordering compound is useful for purposes of thisinvention.

[0248] Suitable solvents include water; diols, such as propylene glycoland glycerol; mono-alcohols, such as ethanol, propanol, and higheralcohols; DMSO; dimethylformamide; N,N-dimethylacetamide; 2-pyrrolidone;N-(2-hydroxyethyl) pyrrolidone, N-methylpyrrolidone,1-dodecylazacycloheptan-2-one and othern-substituted-alkyl-azacycloalkyl-2-ones (azones) and the like.

[0249] U.S. Pat. No. 4,537,776, Cooper, issued Aug. 27, 1985, containsan excellent summary of prior art and background information detailingthe use of certain binary systems for permeant enhancement. Because ofthe completeness of that disclosure, the information and terminologyutilized therein are incorporated herein by reference.

[0250] Similarly, European Patent Application 43,738, referred to above,teaches using selected diols as solvents along with a broad category ofcell-envelope disordering compounds for delivery of lipophilicpharmacologically-active compounds. Because of the detail in disclosingthe cell-envelope disordering compounds and the diols, this disclosureof European Patent Application 43,738 is also incorporated herein byreference.

[0251] A binary system for enhancing metoclopramide penetration isdisclosed in UK Patent Application GB 2,153,223 A, published Aug. 21,1985, and consists of a monovalent alcohol ester of a C8-32 aliphaticmonocarboxylic acid (unsaturated and/or branched if C18-32) or a C6-24aliphatic monoalcohol (unsaturated and/or branched if C14-24) and anN-cyclic compound such as 2-pyrrolidone, N-methylpyrrolidone and thelike.

[0252] Combinations of enhancers consisting of diethylene glycolmonoethyl or monomethyl ether with propylene glycol monolaurate andmethyl laurate are disclosed in U.S. Pat. No. 4,973,468 as enhancing thetransdermal delivery of steroids such as progesterons and estrogens. Adual enhancer consisting of glycerol monolaurate and ethanol for thetransdermal delivery of drugs is shown in U.S. Pat. No. 4,820,720. U.S.Pat. No. 5,006,342 lists numerous enhancers for transdermal drugadministration consisting of fatty acid esters or fatty alcohol ethersof C₂ to C₄ alkanediols, where each fatty acid/alcohol portion of theester/ether is of about 8 to 22 carbon atoms. U.S. Pat. No. 4,863,970shows penetration-enhancing compositions for topical applicationcomprising an active permeant contained in a penetration-enhancingvehicle containing specified amounts of one or more cell-envelopedisordering compounds such as oleic acid, oleyl alcohol, and glycerolesters of oleic acid; a C₂ or C₃ alkanol and an inert diluent such aswater.

[0253] Other chemical enhancers, not necessarily associated with binarysystems include DMSO or aqueous solutions of DMSO such as taught inHerschler, U.S. Pat. No. 3,551,554; Herschler, U.S. Pat. No. 3,711,602;and Herschler, U.S. Pat. No. 3,711,606, and the azones(n-substituted-alkyl-azacycloalkyl-2-ones) such as noted in Cooper, U.S.Pat. No. 4,557,943.

[0254] Some chemical enhancer systems may possess negative side effectssuch as toxicity and skin irritation. U.S. Pat. No. 4,855,298 disclosescompositions for reducing skin irritation caused by chemical enhancercontaining compositions having skin irritation properties with an amountof glycerin sufficient to provide an anti-irritating effect.

[0255] Because the combination of microporation of the stratum corneumand the application of sonic energy accompanied by the use of chemicalenhancers can result in an improved rate of analyte withdrawal orpermeant delivery through the stratum corneum, the specific carriervehicle and particularly the chemical enhancer utilized can be selectedfrom a long list of prior art vehicles some of which are mentioned aboveand incorporated herein by reference. To specifically detail orenumerate that which is readily available in the art is not thoughtnecessary. The invention is not drawn to the use of chemical enhancersper se and it is believed that all chemical enhancers, useful in thedelivery of drugs through the skin, will function with dyes in opticalmicroporation and also with sonic energy in effecting measurablewithdrawal of analytes from beneath and through the skin surface or thedelivery of permeants or drugs through the skin surface.

EXAMPLE 16

[0256] Modulated sonic energy and chemical enhancers were tested fortheir ability to control transdermal flux on human cadaver skin samples.In these tests, the epidermal membrane had been separated from the humancadaver whole skin by the heat-separation method of Example 1. Theepidermal membrane was cut and placed between two halves of thepermeation cell with the stratum corneum facing either the upper (donor)compartment or lower (receiver) compartment. Modified Franz cells wereused to hold the epidermis, as shown in FIG. 2 of U.S. Pat. No.5,445,611. Each Franz cell consists of an upper chamber and a lowerchamber held together with one or more clamps. The lower chamber has asampling port through which materials can be added or removed. A sampleof stratum corneum is held between the upper and lower chambers whenthey are clamped together. The upper chamber of each Franz cell ismodified to allow an ultrasound transducer to be positioned within 1 cmof the stratum corneum membrane. Methylene blue solution was used as anindicator molecule to assess the permeation of the stratum corneum. Avisual record of the process and results of each experiment was obtainedin a time stamped magnetic tape format with a video camera and videocassette recorder (not shown). Additionally, samples were withdrawn formeasurement with an absorption spectrometer to quantitate the amount ofdye which had traversed the stratum corneum membrane during anexperiment. Chemical enhancers suitable for use could vary over a widerange of solvents and/or cell envelope disordering compounds as notedabove. The specific enhancer utilized was:ethanol/glycerol/water/glycerol monooleate/methyl laurate in50/30/15/2.5/2.5 volume ratios. The system for producing and controllingthe sonic energy included a programmable 0-30 MHz arbitrary waveformgenerator (Stanford Research Systems Model DS345), a 20 watt 0-30 MHzamplifier, and two unfocused ultrasound immersion transducers havingpeak resonances at 15 and 25 MHz, respectively. Six cells were preparedsimultaneously for testing of stratum corneum samples from the samedonor. Once the stratum corneum samples were installed, they wereallowed to hydrate with distilled water for at least 6 hours before anytests were done.

EXAMPLE 17

[0257] Effects of Sonic Energy without Chemical Enhancers

[0258] As stated above in Example 16, the heat-separated epidermis wasplaced in the Franz cells with the epidermal side facing up, and thestratum corneum side facing down, unless noted otherwise. The lowerchambers were filled with distilled water, whereas the upper chamberswere filled with concentrated methylene blue solution in distilledwater.

[0259] Heat Separated Epidermis: Immediately after filling the upperchambers with methylene blue solution, sonic energy was applied to oneof the cells with the transducer fully immersed. This orientation wouldcorrespond, for example, to having the transducer on the opposite sideof a fold of skin, or causing the sonic energy to be reflected off areflector plate similarly positioned and being used to “push” analyteout of the other side of the fold into a collection device. The sonicenergy setting was initially set at the nominal operating frequency of25 MHz with an intensity equivalent to a 20 volt peak-to-peak (P-P)input wave form. This corresponds to roughly a 1 watt of average inputpower to the transducer and similarly, assuming the manufacturer'snominal value for conversion efficiency of 1% for this particulartransducer, a sonic output power of around 0.01 watts over the 0.78 cm²surface of the active area or a sonic intensity of 0.13 watts/cm². Threeother control cells had no sonic energy applied to them. After 5 minutesthe sonic energy was turned off. No visual indication of dye flux acrossthe stratum corneum was observed during this interval in any of thecells, indicating levels less than approximately 0.0015% (v/v) of dyesolution in 2 ml of receiver medium.

[0260] Testing of these same 3 control cells and 1 experimental cell wascontinued as follows. The intensity of sonic energy was increased to themaximum possible output available from the driving equipment of a 70volt peak-to-peak input 12 watts average power input or (0.13 watts/cm²)of sonic output intensity. Also, the frequency was set to modulate orsweep from 30 MHz to 10 MHz. This 20 MHz sweep was performed ten timesper second, i.e., a sweep rate of 10 Hz. At these input power levels, itwas necessary to monitor the sonic energy transducer to avoidoverheating. A contact thermocouple was applied to the body of thetransducer and power was cycled on and off to maintain maximumtemperature of the transducer under 42 C. After about 30 minutes ofcycling maximum power at about a 50% duty cycle of 1 minute on and 1minute off, there was still no visually detectable permeation of thestratum corneum by the methylene blue dye.

[0261] A cooling water jacket was then attached to the sonic energytransducer to permit extended excitation at the maximum energy level.Using the same 3 controls and 1 experimental cell, sonic energy wasapplied at maximum power for 12 hours to the experimental cell. Duringthis time the temperature of the fluid in the upper chamber rose to only35 C, only slightly above the approximately 31° C. normal temperature ofthe stratum corneum in vivo. No visual evidence of dye flux through thestratum corneum was apparent in any of the four cells after 12 hrs. ofsonic energy applied as described above.

EXAMPLE 18

[0262] Effects of Sonic Energy without Chemical Enhancers

[0263] Perforated Stratum Corneum: Six cells were prepared as describedabove in Example 16. The clamps holding the upper and lower chambers ofthe Franz cells were tightened greater than the extent required tonormally seal the upper compartment from the lower compartment, and tothe extent to artificially introduce perforations and “pinholes” intothe heat-separated epidermal samples. When dye solution was added to theupper chamber of each cell, there were immediate visual indications ofleakage of dye into the lower chambers through the perforations formedin the stratum corneum. Upon application of sonic energy to cells inwhich the stratum corneum was so perforated with small “pinholes,” arapid increase in the transport of fluid through a pinhole in thestratum corneum was observed. The rate of transport of the indicator dyemolecules was directly related to whether the sonic energy was appliedor not. That is, application of the sonic energy caused an immediate(lag time approximately <0.1 second) pulse of the indicator moleculesthrough the pinholes in the stratum corneum. This pulse of indicatormolecules ceased immediately upon turning off of the sonic energy (ashutoff lag of approximately <0.1 second). The pulse could be repeatedas described.

EXAMPLE 19

[0264] Effects of Sonic Energy and Chemical Enhancers

[0265] Two different chemical enhancer formulations were used. ChemicalEnhancer One or CEI was an admixture of ethanol/glycerol/water/glycerolmonooleate/methyl laurate in a 50/30/15/2.5/2.5 volume ratio. These arecomponents generally regarded as safe, i.e. GRAS, by the FDA for use aspharmaceutical excipients. Chemical Enhancer Two or CE2 is anexperimental formulation shown to be very effective in enhancingtransdermal drug delivery, but generally considered too irritating forlong term transdermal delivery applications. CE2 containedethanol/glycerol/water/lauradone/methyl laurate in the volume ratios50/30/15/2.5/2.5. Lauradone is the lauryl (dodecyl) ester of2-pyrrolidone-5-carboxylic acid (“PCA”) and is also referred to aslauryl PCA.

[0266] Six Franz cells were set up as before (Example 16) except thatthe heat separated epidermis was installed with the epidermal layerdown, i.e., stratum corneum side facing up. Hydration was established byexposing each sample to distilled water overnight. To begin theexperiment, the distilled water in the lower chambers was replaced withmethylene blue dye solution in all six cells. The upper chambers werefilled with distilled water and the cells were observed for about 30minutes confirming no passage of dye to ensure that no pinholeperforations were present in any of the cells. When none were found, thedistilled water in the upper chambers was removed from four of thecells. The other two cells served as distilled water controls. The upperchambers of two of the experimental cells were then filled with CE 1 andthe other two experimental cells were filled with CE2.

[0267] Sonic energy was immediately applied to one of the two CE2 cells.A 25 MHz transducer was used with the frequency sweeping every 0.1second from 10 MHz to 30 MHz at maximum intensity of 0.13 watts/cm².After 10-15 minutes of sonic energy applied at a 50% duty cycle, dyeflux was visually detected. No dye flux was detected in the other fivecells.

[0268] Sonic energy was then applied to one of the two cells containingCE1 at the same settings. Dye began to appear in the upper chamberwithin 5 minutes. Thus, sonic energy together with a chemical enhancersignificantly increased the transdermal flux rate of a marker dyethrough the stratum corneum, as well as reduced the lag time.

EXAMPLE 20

[0269] Effects of Sonic Energy and Chemical Enhancers

[0270] Formulations of the two chemical enhancers, CE1 and CE2, wereprepared minus the glycerin and these new formulations, designated CE1MGand CE2MG, were tested as before. Water was substituted for glycerin sothat the proportions of the other components remained unchanged. Threecells were prepared in modified Franz cells with the epidermal side ofthe heat separated epidermis samples facing toward the upper side of thechambers. These samples were then hydrated in distilled water for 8hours. After the hydration step, the distilled water in the lowerchambers was replaced with either CE1MG or CE2MG and the upper chamberwas filled with the dye solution. Sonic energy was applied to each ofthe three cells sequentially.

[0271] Upon application of pulsed, frequency-modulated sonic energy fora total duration of less than 10 minutes, a significant increase inpermeability of the stratum corneum samples was observed. Thepermeability of the stratum corneum was altered relatively uniformlyacross the area exposed to both the chemical enhancer and sonic energy.No “pinhole” perforations through which the dye could traverse thestratum corneum were observed. The transdermal flux rate was instantlycontrollable by turning the sonic energy on or off. Turning the sonicenergy off appeared to instantly reduce the transdermal flux rate suchthat no dye was visibly being actively transported through the skinsample; presumably the rate was reduced to that of passive diffusion.Turning the sonic energy on again instantly resumed the high level fluxrate. The modulated mode appeared to provide a regular pulsatileincrease in the transdermal flux rate at the modulated rate. When thesonic energy was set to a constant frequency, the maximum increase intransdermal flux rate for this configuration seemed to occur at around27 MHz.

[0272] Having obtained the same results with all three samples, thecells were then drained of all fluids and flushed with distilled wateron both sides of the stratum corneum. The lower chambers were thenimmediately filled with distilled water and the upper chambers wererefilled with dye solution. The cells were observed for 30 minutes. Noholes in the stratum corneum samples were observed and no large amountof dye was detected in the lower chambers. A small amount of dye becamevisible in the lower chambers, probably due to the dye and enhancertrapped in the skin samples from their previous exposures. After anadditional 12 hours, the amount of dye detected was still very small.

EXAMPLE 21

[0273] Effects of Sonic Energy and Chemical Enhancers

[0274] Perforated Stratum Corneum: Three cells were prepared withheat-separated epidermis samples with the epidermal side facing towardthe upper side of the chamber from the same donor as in Example 16. Thesamples were hydrated for 8 hours and then the distilled water in thelower chambers was replaced with either CE1MG or CE2MG. The upperchambers were then filled with dye solution. Pinhole perforations in thestratum corneum samples permitted dye to leak through the stratumcorneum samples into the underlying enhancer containing chambers. Sonicenergy was applied. Immediately upon application of the sonic energy,the dye molecules were rapidly pushed through the pores. As shown above,the rapid flux of the dye through the pores was directly and immediatelycorrelated with the application of the sonic energy.

EXAMPLE 22

[0275] Effects of Sonic Energy and Chemical Enhancers

[0276] A low cost sonic energy transducer, TDK #NB-58S-01 (TDK Corp.),was tested for its capability to enhance transdermal flux rates. Thepeak response of this transducer was determined to be about 5.4 MHz withother local peaks occurring at about 7 MHz, 9 MHz, 12.4 MHz, and 16 MHz.

[0277] This TDK transducer was then tested at 5.4 MHz for its ability toenhance transdermal flux rate in conjunction with CE1MG. Three cellswere set up with the epidermal side facing the lower chamber, then theskin samples were hydrated for 8 hrs. The dye solution was placed in thelower chamber. The transducer was placed in the upper chamber immersedin CE1MG. Using swept frequencies from 5.3 to 5.6 MHz as the sonicenergy excitation, significant quantities of dye moved through thestratum corneum and were detected in the collection well of the cell in5 minutes. Local heating occurred, with the transducer reaching atemperature of 48 C. In a control using CE1MG without sonic energy, a 24hour exposure yielded less dye in the collection well than the 5 minuteexposure with sonic energy.

[0278] This example demonstrates that a low cost, low frequency sonicenergy transducer can strikingly affect transdermal flux rate when usedin conjunction with an appropriate chemical enhancer. Although higherfrequency sonic energy will theoretically concentrate more energy in thestratum corneum, when used with a chemical enhancer, the lower frequencymodulated sonic energy can accelerate the transdermal flux rate to makethe technology useful and practical.

EXAMPLE 23

[0279] Demonstration of molecule migration across human skin: Tests withthe TDK transducer and CE1MG described above were repeated at about 12.4MHz, one of the highest local resonant peaks for the transducer, with afrequency sweep at a 2 Hz rate from 12.5 to 12.8 MHz and an sonic energydensity less than 0.1 W/cm². The epidermal side of the heat-separatedepidermis was facing down, the dye solution was in the lower chamber,and the enhancer solution and the sonic energy were placed in the upperchamber. Within 5 minutes a significant amount of dye had moved acrossthe stratum corneum into the collection well. Ohmic heating in thetransducer was significantly less than with the same transducer beingdriven at 5.4 MHz, causing an increase in temperature of the chemicalenhancer to only about 33 C.

[0280] Even at these low efficiency levels, the results obtained withCE1MG and sonic energy from the TDK transducer were remarkable in themonitoring direction. FIGS. 3A and 3B of U.S. Pat. No. 5,445,611 showplots of data obtained from three separate cells with the transdermalflux rate measured in the monitoring direction. Even at the 5 minutetime point, readily measurable quantities of the dye were present in thechemical enhancer on the outside of the stratum corneum, indicatingtransport from the epidermal side through the stratum corneum to the“outside” area of the skin sample.

[0281] To optimize the use of the sonic energy or the sonicenergy/chemical enhancer approach for collecting and monitoring analytesfrom the body, means for assaying the amount of analyte of interest arerequired. An assay system that takes multiple readings while the unit isin the process of withdrawing analytes by sonic energy with or withoutchemical enhancers makes it unnecessary to standardize across a broadpopulation base and normalize for different skin characteristics andflux rates. By plotting two or more data points in time as the analyteconcentration in the collection system is increasing, a curve-fittingalgorithm can be applied to determine the parameters describing thecurve relating analyte withdrawal or flux rate to the point at whichequilibrium is reached, thereby establishing the measure of the intervalconcentration. The general form of this curve is invariant from oneindividual to another; only the parameters change. Once these parametersare established, solving for the steady state solution (i.e., timeequals infinity) of this function, i.e., when full equilibrium isestablished, provides the concentration of the analyte within the body.Thus, this approach permits measurements to be made to the desired levelof accuracy in the same amount of time for all members of a populationregardless of individual variations in skin permeability.

[0282] Several existing detection techniques currently exist that areadaptable for this application. See, D. A. Christensen, in 1648Proceedings of Fiber Optic, Medical and Fluorescent Sensors andApplications 223-26 (1992). One method involves the use of a pair ofoptical fibers that are positioned close together in an approximatelyparallel manner. One of the fibers is a source fiber, through whichlight energy is conducted. The other fiber is a detection fiberconnected to a photosensitive diode. When light is conducted through thesource fiber, a portion of the light energy, the evanescent wave, ispresent at the surface of the fiber and a portion of this light energyis collected by the detection fiber. The detection fiber conducts thecaptured evanescent wave energy to the photosensitive diode whichmeasures it. The fibers are treated with a binder to attract and bindthe analyte that is to be measured. As analyte molecules bind to thesurface (such as the analyte glucose binding to immobilized lectins suchas concanavalin A, or to immobilized anti-glucose antibodies) the amountof evanescent wave coupling between the two fibers is changed and theamount of energy captured by the detection fiber and measured by thediode is changed as well. Several measurements of detected evanescentwave energy over short periods of time support a rapid determination ofthe parameters describing the equilibrium curve, thus making possiblecalculation of the concentration of the analyte within the body. Theexperimental results showing measurable flux within 5 minutes (FIGS. 3Aand 3B of U.S. Pat. No. 5,445,611) with this system suggest sufficientdata for an accurate final reading are collected within 5 minutes.

[0283] In its most basic embodiment, a device that can be utilized forthe application of sonic energy and collection of analyte comprises anabsorbent pad, either of natural or synthetic material, which serves asa reservoir for the chemical enhancer, if used, and for receiving theanalyte from the skin surface. The pad or reservoir is held in place,either passively or aided by appropriate fastening means, such as astrap or adhesive tape, on the selected area of skin surface.

[0284] An sonic energy transducer is positioned such that the pad orreservoir is between the skin surface and the transducer, and held inplace by appropriate means. A power supply is coupled to the transducerand activated by switch means or any other suitable mechanism. Thetransducer is activated to deliver sonic energy modulated in frequency,phase or intensity, as desired, to deliver the chemical enhancer, ifused, from the reservoir through the skin surface followed by collectionof the analyte from the skin surface into the reservoir. After thedesired fixed or variable time period, the transducer is deactivated.The pad or reservoir, now containing the analyte of interest, can beremoved to quantitate the analyte, for example, by a laboratoryutilizing any number of conventional chemical analyses, or by a portabledevice. Alternately, the mechanism for quantitating the analyte can bebuild into the device used for collection of the analyte, either as anintegral portion of the device or as an attachment. Devices formonitoring an analyte are described in U.S. Pat. No. 5,458,140, which isincorporated herein by reference.

EXAMPLE 24

[0285] An alternate method for detection of an analyte, such as glucose,following the sample collection through the porated skin surface asdescribed above, can be achieved through the use of enzymatic means.Several enzymatic methods exist for the measurement of glucose in abiological sample. One method involves oxidizing glucose in the samplewith glucose oxidase to generate gluconolactone and hydrogen peroxide.In the presence of a colorless chromogen, the hydrogen peroxide is thenconverted by peroxidase to water and a colored product.

[0286] Glucose Oxidase

[0287] Glucose -Gluconolactone+H₂O₂

[0288] 2H₂O₂+chromogen H₂O+colored product

[0289] The intensity of the colored product will be proportional to theamount of glucose in the fluid. This color can be determined through theuse of conventional absorbance or reflectance methods. By calibrationwith known concentrations of glucose, the amount of color can be used todetermine the concentration of glucose in the collected analyte. Bytesting to determine the relationship, one can calculate theconcentration of glucose in the blood of the subject. This informationcan then be used in the same way that the information obtained from ablood glucose test from a finger puncture is used. Results can beavailable within five to ten minutes.

EXAMPLE 25

[0290] Any system using a visual display or readout of glucoseconcentration will indicate to a diagnostician or patient the need foradministration of insulin or other appropriate medication. In criticalcare or other situations where constant monitoring is desired andcorrective action needs to be taken almost concurrently, the display maybe connected with appropriate signal means which triggers theadministration of insulin or other medication in an appropriate manner.For example, there are insulin pumps which are implanted into theperitoneum or other body cavity which can be activated in response toexternal or internal stimuli. Alternatively, utilizing the enhancedtransdermal flux rates possible with micro-poration of the stratumcorneum and other techniques described in this invention, an insulindelivery system could be implemented transdermally, with control of theflux rates modulated by the signal from the glucose sensing system. Inthis manner a complete biomedical control system can be available whichnot only monitors and/or diagnoses a medical need but simultaneouslyprovides corrective action.

[0291] Biomedical control systems of a similar nature could be providedin other situations such as maintaining correct electrolyte balances oradministering analgesics in response to a measured analyte parametersuch as prostaglandins.

EXAMPLE 26

[0292] Similar to audible sound, sonic waves can undergo reflection,refraction, and absorption when they encounter another medium withdissimilar proper-ties [D. Bommannan et al., 9 Pharm. Res. 559 (1992)].Reflectors or lenses may be used to focus or otherwise control thedistribution of sonic energy in a tissue of interest. For many locationson the human body, a fold of flesh can be found to support this system.For example, an earlobe is a convenient location which would allow useof a reflector or lens to assist in exerting directional control (e.g.,“pushing” of analytes or permeants through the porated stratum corneum)similar to what is realized by changing sonic frequency and intensity.

EXAMPLE 27

[0293] Multiple sonic energy transducers may be used to selectivelydirect the direction of transdermal flux through porated stratum corneumeither into the body or from the body. A fold of skin such as an earlobeallow transducers to be located on either side of the fold. Thetransducers may be energized selectively or in a phased fashion toenhance transdermal flux in the desired direction. An array oftransducers or an acoustic circuit may be constructed to use phasedarray concepts, similar to those developed for radar and microwavecommunications systems, to direct and focus the sonic energy into thearea of interest.

EXAMPLE 28

[0294] In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith an excimer laser (e.g. model EMG/200 of Lambda Physik; 193 nmwavelength, 14 ns pulse width) to ablate the stratum corneum accordingto the procedure described in U.S. Pat. No. 4,775,361, herebyincorporated by reference.

EXAMPLE 29

[0295] In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith 1,1′-diethyl-4,4′-carbocyanine iodide (Aldrich, max=703 nm) andthen a total of 70 mJ/cm²/50 ms is delivered to the dye-treated samplewith a model TOLD9150 diode laser (Toshiba America Electronic, 30 mW at690 nm) to ablate the stratum corneum.

EXAMPLE 30

[0296] In this example, the procedure of Example 29 is followed with theexception that the dye is indocyanine green (Sigma cat. no. I-2633;_(max)=775 nm) and the laser is a model Diolite 800-50 (LiCONiX, 50 mWat 780 nm).

EXAMPLE 31

[0297] In this example, the procedure of Example 29 is followed with theexception that the dye is methylene blue and the laser is a modelSDL-8630 (SDL Inc.; 500 mW at 670 nm).

EXAMPLE 32

[0298] In this example, the procedure of Example 29 is followed with theexception that the dye is contained in a solution comprising apermeation enhancer, e.g. CE 1.

EXAMPLE 33

[0299] In this example, the procedure of Example 29 is followed with theexception that the dye and enhancer-containing solution are delivered tothe stratum corneum with the aid of exposure to ultrasound.

EXAMPLE 34

[0300] In this example, the procedure of Example 31 is followed with theexception that the pulsed light source is a short arc lamp emitting overthe broad range of 400 to 1100 nm but having a bandpass filter placed inthe system to limit the output to the wavelength region of about 650 to700 nm.

EXAMPLE 35

[0301] In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first puncturedwith a microlancet (Becton Dickinson) calibrated to produce a microporein the stratum corneum without reaching the underlying tissue.

EXAMPLE 36

[0302] In this example, the procedure of Example 19 is followed with theexception that the heat-separated epidermis samples are first treatedwith focused sonic energy in the range of 70-480 mJ/cm²/50 ms to ablatethe stratum corneum.

EXAMPLE 37

[0303] In this example, the procedure of Example 19 is followed with theexception that the stratum corneum is first punctured hydraulically witha high pressure jet of fluid to form a micropore of up to about 100 μmdiameter.

EXAMPLE 38

[0304] In this example, the procedure of Example 19 is followed with theexception that the stratum corneum is first punctured with short pulsesof electricity to form a micropore of up to about 100 μm diameter.

EXAMPLE 39

[0305] Acoustic Streaming

[0306] A new mechanism and application of sonic energy in the deliveringof therapeutic substances into the body and/or harvesting fluids fromwithin the body into an external reservoir through micro-porationsformed in the biological membrane will now be described. An additionalaspect of this invention is the utilization of sonic energy to create anacoustic streaming effect on the fluids flowing around and between theintact cells in the viable tissues beneath the outer layer of anorganism, such as the epidermis and dermis of the human skin. Acousticstreaming is a well documented mode by which sonic energy can interactwith a fluid medium. Nyborg, Physical Acoustics Principles and Methods,p. 265-331, Vol II-Part B, Academic Press, 1965. The first theoreticalanalysis of acoustic streaming phenomenon was given by Rayleigh (1884,1945). In an extensive treatment of the subject, Longuet-Higgins(1953-1960) has given a result applicable to two dimensional flow thatresults in the near vicinity of any vibrating cylindrical surface. Athree dimensional approximation for an arbitrary surface was developedby Nyborg (1958). As described by Fairbanks et al., 1975 UltrasonicsSymposium Proceedings, IEEE Cat. #75, CHO 994-4SU, sonic energy, and theacoustic streaming phenomenon can be of great utility in acceleratingthe flux of a fluid through a porous medium, showing measurableincreases in the flux rates by up to 50 times that possible passively orwith only pressure gradients being applied.

[0307] All previous transdermal delivery or extraction efforts utilizingultrasound have focused on methods of interaction between the sonicenergy and the skin tissues designed to permeabilize the stratum corneumlayer. The exact mode of interaction involved has been hypothesized tobe due exclusively to the local elevation of the temperature in the SClayer, and the resultant melting of the lipid domains in theintercellular spaces between the corneocytes. Srinivasan et al. Otherresearchers have suggested that micro-cavitations and or shearing of thestructures in the stratum corneum opens up channels through which fluidsmay flow more readily. In general, the design of the sonic systems forthe enhancement of transdermal flux rates has been based on the earlyrealization that the application of an existing therapeutic ultrasoundunit designed to produce a “deep-heating” effect on the subject, whenused in conjunction with a topical application of a gelled or liquidpreparation containing the drug to be delivered into the body, couldproduce a quantifiable increase in the flux rate of the drug into thebody. In the context of the method taught herein to create micropores inthis biological membrane, the use of sonic energy may now be thought ofin a totally new and different sense than the classically definedconcepts of sonophoresis.

[0308] Based on the experimental discovery mentioned in U.S. Pat. Nos.5,458,140 and 5,445,611 that when a small hole existed or was created inthe stratum corneum (SC) in the Franz cells used in the in vitrostudies, that the application of an appropriately driven ultrasonictransducer to the fluid reservoir on either side of the porated SCsample, an “acoustic streaming” event could be generated wherein largeflux rates of fluid where capable of being pumped through this poratedmembrane.

[0309] With the method taught herein to create the controlledmicro-porations in the biological membrane in the organism, theapplication of the fluid streaming mode of sonic/fluid interaction tothe induction of fluid into or out of the organism may now bepractically explored. For example, clinical studies have shown that bymaking a series of four 80 μm diameter micropores in a 400 μm square,and then applying a mild (10 to 12 inches of Hg) suction to this area,an average of about 1 μl of interstitial fluid can be induced to leavethe body for external collection in an external chamber. By adding asmall, low power sonic transducer to this system, configured such thatit actively generates inwardly converging concentric circular pressurewaves in the 2 to 6 mm of tissue surrounding the poration site, it hasbeen demonstrated that this ISF flux rate can be increased by 50%.

[0310] By relieving ourselves of the desire to create some form ofdirect absorption of sonic energy in the skin tissues (as required togenerate heating), frequencies of sonic energy can be determined forwhich the skin tissues are virtually transparent, that is at the verylow frequency region of 1 kHz to 500 KHz. Even at some of the lowestfrequencies tested, significant acoustic streaming effects could beobserved by using a micro-scope to watch an in vivo test wherein thesubject's skin was micro-porated and ISF was induced to exit the body anpool on the surface of the skin. Energizing the sonic transducer showeddramatic visual indications of the amount of acoustic streaming as smallpieces of particulate matter were carried along with the ISF as itswirled about. Typical magnitude of motion exhibited can be described asfollows: for a 3 mm diameter circular pool of ISF on the surface of theskin, a single visual particle could be seen to be completing roughly 3complete orbits per second. This equates to a linear fluid velocity ofmore than 2.5 mm/second. All of this action was demonstrated with sonicpower levels into the tissues of less than 100 mW/cm2.

[0311] While one can easily view the top surface of the skin, and thefluidic activity thereon, assessing what is taking place dynamicallywithin the skin tissue layers in response to the coupling into thesetissues of sonic energy is much more difficult. One can assume, that ifsuch large fluid velocities (e.g. >2.5 mm/S) may be so easily induced onthe surface, then some noticeable increase in the fluid flow in theintercellular channels present in the viable dermal tissues could alsobe realized in response to this sonic energy input. Currently, anincrease in harvested ISF through a given set of microporations when alow frequency sonic energy was applied to the area in a circlesurrounding the poration sites has been quantified. In this experiment,an ISF harvesting technique based solely on a mild suction (10 to 12inches of HG) was alternated with using the exact same apparatus, butwith the sonic transducer engaged. Over a series of 10 two-minuteharvesting periods, five with mere suction and five with both suctionand sonic energy active, it was observed that by activating the sonicsource roughly 50% more ISF was collectable in the same time period.These data are shown in FIG. 30. This increase in ISF flux rate wasrealized with no reported increase in sensation from the test subjectdue to the sonic energy. The apparatus used for this experiment isillustrated in FIGS. 31-33. The transducer assembly in FIGS. 31-33 iscomprised of a thick walled cylinder of piezo-electric material, with aninternal diameter of roughly 8 mm and a wall thickness of 4 mm. Thecylinder has been polarized such that when an electrical field isapplied across the metallized surfaces of the outer diameter and innerdiameter, the thickness of the wall of the cylinder expands or contractsin response to the field polarity. In practice, this configurationresults in a device which rapidly squeezes the tissue which has beensuctioned into the central hole, causing an inward radial acousticstreaming effect on those fluids present in these tissues. This inwardacoustic streaming is responsible for bringing more ISF to the locationof the micro-porations in the center of the hole, where it can leave thebody for external collection.

[0312] A similar device shown in FIG. 34A-B was built and tested andproduced similar initial results. In the FIG. 34A-B version, anultrasonic transducer built by Zevex, Inc. Salt Lake City, Utah, wasmodified by having a spatulate extension added to the sonic horn. A 4 mmhole was placed in the 0.5 mm thick spatulate end of this extension.When activated, the principle motion is longitudinal along the length ofthe spatula, resulting in essentially a rapid back and forth motion. Thephysical perturbation of the metallic spatula caused by the placement ofthe 4 mm hole, results in a very active, but chaotic, large displacementbehavior at this point. In use, the skin of the subject was suctioned upinto this hole, and the sonic energy was then conducted into the skin ina fashion similar to that illustrated in FIG. 33.

[0313] The novel aspect of this new application of ultrasound lies inthe following basic areas:

[0314] 1. The function of the sonic energy is no longer needed to befocused on permeabilizing the SC barrier membrane as taught by Langer,Kost, Bommannan and others.

[0315] 2. A much lower frequency system can be utilized which has verylittle absorption in the skin tissues, yet can still create the fluidicstreaming phenomenon desired within the intercellular passagewaysbetween the epidermal cells which contain the interstitial fluid.

[0316] 3. The mode of interaction with the tissues and fluids therein,is the so-called “streaming” mode, recognized in the sonic literature asa unique and different mode than the classical vibrational interactionscapable of shearing cell membranes and accelerating the passivediffusion process.

[0317] By optimizing the geometric configuration, frequency, power andmodulations applied to the sonic transducer, it has been shown thatsignificant increases in the fluid flux through the porated skin sitescan be achieved. The optimization of these parameters is designed toexploit the non-linearities governing the fluid flow relationships inthis microscopically scaled environment. Using frequencies under 200kHz, large fluidic effects can be observed, without any detectableheating or other negative tissue interactions. The sonic power levelsrequired to produce these measurable effects are very low, with averagepower levels typically under 100 milliwatts/cm2.

[0318] Therefore, the above examples are but representative of systemswhich may be employed in the utilization of sonic energy or sonic energyand chemical enhancers in the collection and quantification of analytesfor diagnostic purposes and for the transmembrane delivery of permeants.The invention is directed to the discovery that the poration of thebiological membrane followed by the proper use of sonic energy,particularly when accompanied with the use of chemical enhancers,enables the noninvasive or minimally invasive transmembranedetermination of analytes or delivery of permeants. However, theinvention is not limited only to the specific illustrations. There arenumerous poration techniques and enhancer systems, some of which mayfunction better than another, for detection and withdrawn of certainanalytes or delivery of permeants through the stratum corneum. However,within the guidelines presented herein, a certain amount ofexperimentation to obtain optimal poration, enhancers, or optimal time,intensity and frequency of applied sonic energy, as well as modulationof frequency, amplitude and phase of applied sonic energy can be readilycarried out by those skilled in the art. Therefore, the invention islimited in scope only by the following claims and functional equivalentsthereof.

[0319] Further Advancements and Improvements

[0320] Advancements and improvements to the microporation techniqueshave been made, particularly suitable for, though not limited to,delivery applications. One advancement is to porate, using any one ofthe aforementioned microporation techniques, to a selected depth into orthrough biological membranes, including the skin, the mucous membrane,or plant outer layer, particularly for delivery of a drug or bioactiveagent into the body. Another advancement is to deliver bioactive agentsinto the organism through micropores formed in the biological membrane.Still another advancement is to apply permeation enhancement measuresbefore, during, or after microporation, so as to increase thepermeability of layers within the microporated skin or mucosa whendelivering substances, such as drugs or bioactive agents, thereinto ortherethrough.

[0321] The micropore formed in the biological membrane may extend to aselected depth. A micropore extending into the epidermis may penetrateonly the stratum corneum or selected depths into the viable cell layeror underlying connective tissue layer. Similarly, if formed in themucous membrane, the micropore may penetrate only the superficial partof the epithelial layer or selected depths into the epithelial lining orunderlying lamina propria and into tissue beneath. The micropore depthin either case can extend through the entire depth of the biologicalmembrane.

[0322] As an example for microporating to a selected depth, if oneutilizes a heat probe which can continue to deliver sufficient energyinto or through the fully hydrated viable cell layers beneath thestratum corneum, the poration process can continue into the body toselected depths, penetrating through the epidermis, the dermis, and intoor through the subcutaneous layers below if desired. The concern when asystem is designed to create a micropore extending some distance into orthrough the viable tissues in the epidermis or dermis, or the epitheliallining or lamina propria, is how to minimize damage to the adjacenttissue and the sensation to the subject during the poration process.

[0323] Experimentally, we have shown that if the heat probe used is asolid, electrically or optically heated element, with the active heatedprobe tip physically defined to be no more than a few hundred micronsacross and protruding up to a few millimeters from the supporting base,that a single pulse, or multiple pulses of current can deliver enoughthermal energy into the tissue to allow the ablation to penetrate asdeep as the physical design allows, that is, until the support baselimits the extent of the penetration into or through the tissue. If theelectrical and thermal properties of said heat probe, when it is incontact with the tissues, allow the energy pulse to modulate thetemperature of said probe rapidly enough, this type of deep tissueporation can be accomplished with essentially no pain to the subject.Experiments have shown that if the required amount of thermal energy isdelivered to the probe within less than roughly 20 milliseconds (20-50msec), that the procedure is painless. Conversely, if the energy pulsemust be extended beyond roughly 20 milliseconds (20-50 msec), thesensation to the subject increases rapidly and non-linearly as the pulsewidth is extended.

[0324] Similarly, an electrically heated probe design which supportsthis type of selected deep poration can be built by bending a 50 to 150micron diameter tungsten wire into a sharp kink, forming a close to 180degree bend with a minimal internal radius at this point. This miniature‘V’ shaped piece of wire can then be mounted such that this ‘V’ extendssome distance out from a support piece which has copper electrodesdeposited upon it. The distance to which the wire extends out from thesupport will define the maximum penetration distance into the tissuewhen the wire is heated. Each end of the tungsten ‘V’ will be attachedto one of the electrodes on the support carrier which in turn can beconnected to the current pulsing circuit. When the current is deliveredto the wire in an appropriately controlled fashion, the wire willrapidly heat up to the desired temperature to effect the thermalablation process in a single pulse or in multiple pulses of current. Bymonitoring the dynamic impedance of the probe and knowing thecoefficient of resistance versus temperature of the tungsten element,closed loop control of the temperature of the contact point can easilybe established. Also, by dynamically monitoring the impedance throughthe body from the contact point of the probe and a second electrodeplaced some distance away, the depth of the pore can be determined basedon the different impedance properties of the tissue as one penetratesdeeper into the body. Once the impedance properties of a selected tissueof a selected organism have been routinely determined, this parametercan be used to determine the pore depth and can be used in a controlsystem to control pore depth.

[0325] Likewise, one embodiment of an optically heated probe designwhich supports this type of selected depth poration can be built bytaking an optical fiber and placing on one end a tip comprised of asolid cap or coating. A light source such as a laser diode will becoupled into the other end of the fiber. The side of tip facing thefiber must have a high enough absorption coefficient over the range ofwavelengths emitted by the light source that when the photons reach theend of the fiber and strike this face, some of them will be absorbed andsubsequently cause the tip to heat up. The specific design of this tip,fiber and source assembly may vary widely, however fibers with grossdiameters of 50 to 1000 microns across are common place items today andsources emitting up to thousands of watts of optical energy aresimilarly common place. The tip forming the actual heat probe can befabricated from a high melting point material, such as tungsten andattached to the fiber by machining it to allow the insertion of thefiber into a cylindrical bore at the fiber end. If the distal end of thetip has been fabricated to limit the thermal diffusion away from thistip and back up the supporting cylinder attaching the tip to the fiberwithin the time frame of the optical pulse widths used, the photonsincident upon this tip will elevate the temperature rapidly on both thefiber side and the contact side which is placed against the tissuessurface. The positioning of the fiber/tip assembly onto the tissuesurface, can be accomplished with a simple mechanism designed to holdthe tip against the surface under some spring tension such that as thetissue beneath it is ablated, the tip itself will advance into thetissue. This allows the thermal ablation process to continue into orthrough the tissue as far as one desires. An additional feature of thisoptically heated probe design is that by monitoring the black bodyradiated energy from the heated tip that is collected by the fiber, avery simple closed loop control of the tip temperature can be effected.Also, as described earlier, by dynamically monitoring the impedancethrough the body from the contact point of the probe and a secondelectrode placed some distance away, the depth of the pore can beestimated based on the different impedance properties of the tissue asone penetrates deeper into the body. The relationship between pulsewidth and sensation for this design is essentially the same as for theelectrically heated probe described earlier.

[0326] For example, some vaccine applications are known to be mosteffective if delivered into the dermal layer so as to be in proximity tothe Langerhan's or dendritic cells or other cells important for thisimmune response. This would imply a poration depth designed to passthrough the epidermis, which in most cases would be roughly 180 micronsto 250 microns deep.

[0327] As another example, when delivering some proteins and peptides,it is desirable to minimize the immune response to the permeant at thesite of the administration and at the same time bypass the proteaseactive zones in the skin tissues. In this case an even deeper pore maybe desired, going as deep as 300 microns into the skin.

[0328] Alternatively, it may be desirable to leave a minimally thicklayer of intact stratum corneum to minimize rapid initial uptake of apermeant and to provide some retention of the stratum corneum's barrierfunction to provide for a controlled release over a longer period oftime.

[0329] An additional feature of this invention is the large increase inefficiency which can be gained by combining the poration of the layersof the biological membrane with other permeation enhancement techniqueswhich can now be optimized to function on the various barriers to effectdelivery of the desired compound into the internal spaces as necessaryfor bio-effectivity. In particular, if one is delivering a nucleic acidcompound either naked, fragmented, encapsulated or coupled to anotheragent, it is often desired to get the nucleic acid into the living cellswithout killing the cell to allow the desired uptake and subsequentperformance of the therapy. The application of electroporation,iontophoresis, magnetic fields and thermal and sonic energy can causeopenings to form, temporarily, in the cell membranes and other internaltissues. Because we have shown how to breach the stratum corneum orepithelial layer of the mucosal membrane or the outer layer of a plant,and if desired the epidermis and dermis or deeper into a plant,electroporation, iontophoresis, magnetic fields and thermal and sonicenergy can now be used with parameters that can be tailored to actselectively on these underlying tissue barriers and permeabilize thecell, capillary or other membranes within the targeted tissue.Electroporation, iontophoresis, magnetic fields, and thermal and sonicenergy were previously inapplicable for this use.

[0330] In the case of electroporation, where pulses exceeding 50 to 150volts are routinely used to electroporate the stratum corneum or outerlayer of the mucosal membrane or outer layer of a plant, in theenvironment we present, pulses of only a few volts or less aresufficient to electroporate the cell, capillary or other membraneswithin the targeted tissue. This is principally due to the dramaticreduction in the number of insulating layers present between theelectrodes once the skin, mucosal layer, or outer layer of a plant hasbeen opened.

[0331] Similarly, iontophoresis can be shown to be effective to modulatethe flux of a fluid media containing the nucleic acid through themicropores with very small amounts of current due to the dramaticreduction in the physical impedance to fluid flow through these poratedlayers.

[0332] In the case of sonic energy, whereas classically sonic energy hasbeen used to accelerate the permeation of the stratum corneum or mucosallayer, by eliminating this barrier, sonic energy can now be used topermeabilize the cell, capillary or other membranes within the targetedtissue. As in the cases of electroporation and iontophoresis, we havedemonstrated that the sonic energy levels needed to effect a notableimprovement in the transmembrane flux of a substance are much lower thanwhen skin or mucosal layers are left intact. Other permeationenhancement measures involve changing the osmotic pressure or physicalpressure at the microporated site, for example applying a mild pneumaticpressure to the permeant reservoir to force a particular fluid flow intothe organism through the micropores

[0333] The mode of operation of all of these active methods,electroporation, iontophoresis, magnetic or thermal or sonic energy,when applied solely or in combination, after the poration of the skin ormucosal layer or the outer layer of a plant has been effected, has theadvantage of being able to use parameters typically used in in vitroapplications where single cell membranes are opened up for the deliveryof a substance. Examples of these parameters are well known in theliterature. For example, Sambvrook et al., Molecular Cloning: ALaboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989.

[0334] The micropores produced in the biological membrane by the methodsof the present invention allow high flux rates of large (as well assmall) molecular weight therapeutic compounds to be deliveredtransdermally or transmucosally or transmembrane. In addition, thesenon-traumatic microscopic openings into the body allow access to variousanalytes within the body, which can be assayed to determine theirinternal concentrations.

[0335] Delivery of Bioactive Agents

[0336] Still another advancement of the present invention involves theuse of poration of the biological membrane for the delivery of abioactive agent, e.g., polypeptides, including proteins and peptides(e.g., insulin); releasing factors; including LHRH; carbohydrates (e.g.,heparin); nucleic acids; vaccines; and pharmacologically active agentssuch as antiinfectives such as antibiotics and antiviral agents;analgesics and analgesic combinations; anorexics; antihelminthics;antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants;antidiabetic agents; antidiarrheals; antihistamines; antiinflammatoryagents; antimigraine preparations; antinauseants; antineoplastics;antiparkinsonism drugs; antipruritics; antipsychotics; antipyretics;antispasmodics; anticholinergics; sympathomimetics; xanthinederivatives; cardiovascular preparations including potassium and calciumchannel blockers, beta-blockers, alpha-blockers, and antiarrhythmics;antihypertensives; diuretics and antidiuretics; vasodilators includinggeneral coronary, peripheral and cerebral; central nervous systemstimulants; vasoconstrictors; cough and cold preparations, includingdecongestants; hormones such as estradiol, testosterone, progesteroneand other steroids and derivatives and analogs, includingcorticosteroids; hypnotics; immunosuppressives; muscle relaxants;parasympatholytics; psychostimulants; sedatives; and tranquilizers. Bythe method of the present invention, both ionized and nonionized drugsmay be delivered, as can drugs of either high, medium or low molecularweight.

[0337] Delivery of DNA and/or RNA can be used to achieve expression of apolypeptide, stimulate an immune response, or to inhibit expression of apolypeptide through the use of an “antisense” nucleic acid, especiallyan antisense RNA. The term “polypeptide” is used herein without anyparticular intended size limitation, unless a particular size isotherwise stated, and includes peptides of any length includingproteins. Typical of polypeptides that can be expressed are thoseselected from the group consisting of oxytocin, vasopressin,adrenocorticotrophic hormone, epidermal growth factor, prolactin,luteinizing hormone releasing hormone, growth hormone, growth hormonereleasing factor, insulin-like growth factors, insulin, erythropoietin,obesity protein such as leptin, somatostatin, glucagon, glucagon-likeinsulinotropic factors, parathyroid hormone, interferon, gastrin,interleukin-2 and other interleukins and lymphokines, tetragastrin,pentagastrin, urogastroine, secretin, calcitonin, enkephalins,endorphins, angiotensins, renin, bradykinin, bacitracins, polymixins,colistins, tyrocidin, gramicidines, and synthetic analogues,modifications and pharmacologically active fragments thereof, monoclonalantibodies and vaccines. This group is not to be considered limiting;the only limitation to the peptide or protein drug that may be expressedis one of functionality. Delivery of DNA and/or RNA is useful in genetherapy, vaccination, and any therapeutic situation in which a nucleicacid or a polypeptide should be administered in vivo. E.g., U.S. Pat.No. 5,580,859, hereby incorporated by reference.

[0338] One illustrative embodiment of the invention is a method forobtaining long term administration of a polypeptide comprising poratingthe biological membrane and then delivering a DNA encoding thepolypeptide through the pores in the biological membrane, whereby cellsof the tissue take up the DNA and produce the polypeptide for at leastone month, and more preferably at least 6 months. Another illustrativeembodiment of the invention is a method for obtaining transitoryexpression of a polypeptide comprising porating the biological membraneand then delivering an RNA or DNA encoding the polypeptide through thepores of the biological membrane, whereby cells of the tissue (e.g., theskin, mucous membrane, capillaries, or underlying tissue) take up theRNA or DNA and produce the polypeptide for less than about 20 days,usually less than about 10 days, and often less than about 3-5 days. Thecells which take up the RNA or DNA could include the cells of thebiological membrane, the underlying tissue or other target tissuereached by way of the capillaries.

[0339] The DNA and/or RNA can be naked nucleic acid optionally in acarrier or vehicle, and/or can be contained within microspheres,liposomes and/or associated with transfection-facilitating proteins,microparticles, lipid complexes, viral particles, charged or neutrallipids, carbohydrates, calcium phosphate or other precipitating agents,and/or other substances for stabilizing the nucleic acid. The nucleicacid can be contained in a viral vector that either integrates into thechromosome or is nonintegrating, in a plasmid, or as a nakedpolynucleotide. The nucleic acid can encode a polypeptide, oralternatively can code for an antisense RNA, for example for inhibitingtranslation of a selected polypeptide in a cell. When the nucleic acidis DNA, it can be a DNA sequence that is itself non-replicating, but isinserted into a plasmid wherein the plasmid further comprises areplicator. The DNA may also contain a transcriptional promoter, such asthe CMV IEP promoter, which is functional in humans. The DNA can alsoencode a polymerase for transcribing the DNA. In one preferredembodiment, the DNA codes for both a polypeptide and a polymerase fortranscribing the DNA. The DNA can be delivered together with thepolymerase or with mRNA coding therefor, which mRNA is translated in thecell. In this embodiment, the DNA is preferably a plasmid, and thepolymerase is preferably a phage polymerase, such as the T7 polymerase,wherein the T7 polymerase gene should include a T7 promoter.

[0340] The method can be used to treat a disease associated with adeficiency or absence or mutation of a specific polypeptide. Inaccordance with another aspect of the invention, the method provides forimmunizing an individual, wherein such individual can be a human or ananimal, comprising delivering a DNA and/or RNA to the individual whereinthe DNA and/or RNA codes for an immunogenic translation product thatelicits an immune response against the immunogen. The method can be usedto elicit a humoral immune response, a cellular immune response, or amixture thereof.

EXAMPLE 40

[0341] This illustrative example shows the preparation and delivery ofan mRNA.

[0342] In general, it should be apparent that, in practicing theinvention, a suitable plasmid for in vitro transcription of mRNA can bereadily constructed by those of ordinary skill in the art with avirtually unlimited number of cDNAs. Such plasmids can advantageouslycomprise a promoter for a selected RNA polymerase, followed by a 5′untranslated region, a 3′ untranslated region, and a template for apolyadenylate tract. There should be a unique restriction site betweenthese 5′ and 3′ untranslated regions to facilitate the insertion of anyselected cDNA into the plasmid. Then, after cloning the plasmidcontaining the selected gene, the plasmid is linearized by digestion inthe polyadenylation region and is transcribed in vitro to form mRNAtranscripts. These transcripts are preferably provided with a 5′ cap.Alternatively, a 5′ untranslated sequence such as EMC can be used, whichdoes not require a 5′ cap.

[0343] The readily available SP6 cloning vector, pSP64T, provides 5′ and3′ flanking regions from the Xenopus-globin gene, an efficientlytranslated mRNA. Any cDNA containing an initiation codon can beintroduced into this plasmid, and mRNA can be prepared from theresulting template DNA. This particular plasmid can be digested withBglII to insert any selected cDNA coding for a polypeptide of interest.Although good results can be obtained with pSP64T when linearized andthen transcribed with SP6 RNA polymerase, it is preferable to use theXenopus-globin flanking sequences of pSP64T with the phage T7 RNApolymerase. This is accomplished by purifying an approximately 150 bpHindIII/EcoRI fragment from pSP64T and inserting it into a linearizedapproximately 2.9 kb HindIII/EcoRI fragment of pIBI131 (commerciallyavailable from International Biotechnologies, Inc., New Haven, Conn.)with T4 ligase. The resulting plasmid, pXBG, is adapted to receive anygene of interest at a unique BglII site situated between the twoXenopus-globin sequences and for transcription of the selected gene withT7 polymerase.

[0344] A convenient marker gene for demonstrating in vivo expression ofexogenous polynucleotides is chloramphenicol acetyltransferase, CAT. TheCAT gene from the small BamHI/HindIII fragment of pSV2-CAT (ATCC No.37155) and the BglII-digested pXBG are both incubated with the Klenowfragment of E. coli DNA polymerase to generate blunt ends, and then areligated with T4 DNA ligase to form pSP-CAT. This plasmid is thendigested with PstI and HindIII and the small fragment, comprising theCAT gene between the 5′ and 3′-globin flanking sequences of pSP64T. TheT7 promoter-containing plasmid pIBI131 is also digested with PstI andHindIII, and the long fragment is purified. This fragment is thenligated to the CAT gene containing fragment with T4 DNA ligase to formthe plasmid pT7CAT-An.

[0345] The pT7CAT-An plasmid DNA is purified according to methods wellknown in the art, e.g. U.S. Pat. No. 5,580,859. The resulting purifiedplasmid DNA is then linearized downstream of the polyadenylate regionwith an excess of PstI, and the resulting linearized DNA is thenpurified and transcribed in vitro according to the method of Example 5of U.S. Pat. No. 5,580,859. The resulting mRNA is then purifiedaccording to the method of Example 5 of U.S. Pat. No. 5,580,859, whichis sufficiently pure for delivery according the present invention.

[0346] The purified mRNA is delivered by porating a selected site on anindividual according to the microporation procedures with selected poredepth which optimizes bioactivity and delivering an effective amount ofmRNA to such site such that the mRNA passes through the skin or mucousmembrane into the underlying tissue, where the mRNA is taken up by thecells. This delivery through the porated stratum corneum or mucousmembrane can be aided with sonic energy and/or use sonic energyaccording to the procedure of Example 15 and/or with electroporation toenhance cellular uptake, and/or with a pressure differential forinducing flux through the pores in the skin or mucous membrane.Moreover, delivery can be aided by placing the mRNA is a carriersolution, such as a positively charged lipid complex or liposome, forenhancing the diffusion of the mRNA through the pores into the body orfor facilitating uptake of the mRNA into cells.

EXAMPLE 41

[0347] This example shows immunization of an individual with mRNAencoding the gp120 protein of HIV. The mRNA is prepared according to theprocedure of Example 40 except the gene for gp120 (pIIIenv3-1 from theAIDS Research and Reagent Program, National Institute of Allergy andInfectious Disease, Rockville, Md.) is inserted into the plasmid pXBG ofExample 40. The mRNA containing the gp120 gene is delivered according tothe procedure of Example 40.

EXAMPLE 42

[0348] This example shows immunization of an individual with DNAencoding the gp120 protein of HIV. The gp120 gene is inserted into arecombinant adenovirus according to the procedure of P. Muzzin et al.,Correction of Obesity and Diabetes in Genetically Obese Mice by LeptinGene Therapy, 93 Proc. Nat'l Acad. Sci. USA 14804-14808 (1996); G. Chenet al., Disappearance of Body Fat in Normal Rats Induced byAdenovirus-mediated Leptin Gene Therapy, 93 Proc. Nat'l Acad. Sci. USA14795-99 (1996), hereby incorporated by reference. The resulting DNA isdelivered according to the procedure of Example 41.

EXAMPLE 43

[0349] In this example, the procedure of Example 42 is followed exceptthat DNA encoding glycoprotein D of HSV-2 is substituted for the DNAencoding gp120 protein and additionally is combined with an effectiveamount of the glycoprotein D.

EXAMPLE 44

[0350] In this example, a nucleic acid encoding the obesity proteinleptin, such as a human leptin or a rat leptin cDNA, C. Guoxun et al.,Disappearance of Body Fat in Normal Rats Induced by Adenovirus-mediatedLeptin, 93 Proc. Nat'l Acad. Sci. USA 14795-99 (1996), or a mouse leptincDNA, P. Muzzin et al., Correction of Obesity and Diabetes inGenetically Obese Mice by Leptin Gene Therapy, 93 Proc. Nat'l Acad. Sci.USA 14804-14808 (1996), both of which are hereby incorporated byreference, is delivered in an appropriate plasmid vector. The mammalianexpression vector, pEUK-CI (Clontech, Palo Alto, Calif.) is designed fortransient expression of cloned genes. This vector is a 4.9 kb plasmidcomprising a pBR322 origin of replication and an ampicillin resistancemarker for propagation in bacteria, and also comprising the SV40 originof replication, SV40 late promoter, and SV40 late polyadenylation signalfor replication and expression of a selected gene in a mammalian cell.Located between the SV40 late promoter and SV40 late polyadenylationsignal is a multiple cloning site (MCS) of unique XhoI, XbaI, SmaI,SacI, and BamHI restriction sites. DNA fragments cloned into the MCS aretranscribed into RNA from the SV40 late promoter and are translated fromthe first ATG codon in the cloned fragments. Transcripts of cloned DNAare spliced and polyadenylated using the SV40 VPI processing signals.The leptin gene is cloned into the MCS of pEUK-C1 using techniques wellknown in the art, e.g. J. Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., 1989), hereby incorporated by reference. Theresulting plasmid is delivered to a human or animal individual afterporation of the skin or mucosal membrane according to the proceduredescribed above in the previous examples.

EXAMPLE 45

[0351] Delivery of Heparin. Heparins are useful therapeutic substanceswherein the maintenance of a basal level equivalent to an intravenousinfusion of roughly 1000 to 5000 IU per hour, subcutaneous injectionstwice daily of 5000-1000 IU of heparin, or 1500-6000 IU of low molecularweight heparins is a typical clinical dosage. Normally, heparin wouldnot be considered a good candidate for a transdermal delivery systembecause of its relatively high resistance to crossing the skin duemainly to the molecular weight, 5000 to 30000 Da, of the substance. Withthe microporation techniques disclosed herein, a significant flux rateof heparin was easily achieved when a sufficient quantity of heparin,such as from a delivery reservoir attached to the skin surface where themicropores were placed, was administered. A heparin solution was appliedto skin porated to a depth of approximately 100 μm, allowing eitherpassive diffusion or coupled with iontophoresis (about 1 mA/cm²) thatwas applied for a sufficient period of time to transport the heparinthrough the micropores into the underlying tissues. Evidence of deliveryof heparin was observed by increased capillary dilation and permeabilityas evidenced by microscopic examination of the in vivo site for both thepassive and iontophoretically enhanced delivery. In addition to showinga significant heparin flux using passive diffusion as the main drivingforce, heparin, being a highly charged compound, is a natural candidatefor the coupling of an electrical field with the micropores to allow foran actively controllable flux rate and higher flux rates than possiblethrough the same number of micropores than is possible with the passivediffusion method. An experiment was conducted wherein a site on thevolar forearm of a healthy male volunteer was prepared by creating amatrix of 36 micropores within a 1 square cm area. A small reservoircontaining a sodium heparin solution and the negative electrode for aniontophoretic system was attached to the site. The positive electrodewas attached to the subject's skin some distance away using a hydrogelelectrode obtained from Iomed, a commercial supplier of iontophoreticsystems. The system was run for ten minutes at 0.2 milliamperes persquare cm. After this period, microscopic examination of the site showeddirect evidence of the delivery of heparin from the vasodilation of thecapillaries and when a suction force was applied to extract a sample ofinterstitial fluid from the micropores, enough red blood cells exitedthe capillaries under this force to tint the collected ISF pink,indicating increased vaso-permeability in the area. Furthermore, whenplaced aside to see if the red cells would clot, no clotting took place,indicating the anticlotting effect of the heparin present in the tissuesat work.

EXAMPLE 46

[0352] Delivery of Insulin: Insulin, like many compounds normallypresent in the healthy individual, is a polypeptide which must bemaintained in individuals, such as diabetics who need exogenous insulin,at both a basal level and be given in a pulsatile bolus fashion inresponse to meals and the subject's activity levels. Currently this isachieved via subcutaneous injections of fast acting and slow actingformulations. Because of the molecular weight of insulin, typically6000, it is not able to be delivered at clinically useful levels withtraditional transdermal or transmucosal methods. However, by opening themicropores through the barrier layers of the skin or mucosa, a clearpath is provided allowing the delivery of the insulin into the viabletissues wherein the interstitial fluid present in these tissues willallow diffusion (including osmotically driven) of the insulin to andinto the lymph system and capillary bed, delivering clinically usefulamounts. A concentrated insulin solution containing 3500 IU/ml ofrecombinant human insulin purchased from Boehringer-Mannhein Co., wasapplied in a reservoir to a porated area of the subject's skin on thevolar forearm covering 4 square cm. The healthy, 44 year old, male,non-diabetic, subject fasted for 14 hours prior to the start of theexperiment. Intravenous and finger stick blood samples were drawnperiodically prior to and after the delivery phase began and assayed forglucose, insulin and C-peptide. The finger stick blood glucose datashowed a significant and rapid depression of the subject's glucoselevels after approximately 4 hours, dropping from 100 mg/dl at the startto 67 mg/dl over a ten minute cycle and then returning to 100 in anadditional ten minutes, hypothesized to be due to the subject'scounter-regulatory system engaging and compensating for the deliveredinsulin. A repeat of this procedure with the addition of ultrasoundoperating at 44 khz, and 0.2 watts/square cm indicated a more rapiddelivery of the insulin as evidenced by the subject's glucose levelswhich dropped from 109 mg/dl to 78 mg/dl less than 30 minutes after thedelivery began. As in the case of example 45, for heparin delivery, alow current iontophoretic system can be coupled with the micropores tofacilitate a greater flux rate and provide the ability to modulate thisflux rate by varying the current, allowing a delivery on demand type ofsystem to be built. Previous work with insulin has typically shown thatrelatively high iontophoretic currents are required to overcome thestrong barrier properties of the intact stratum corneum. By porating thestratum corneum or mucosa, and optionally setting the porationparameters to make a deeper pore into or through the targeted biologicalmembrane, a lower current density is required to produce the desiredinsulin flux rates.

[0353] Similarly, for uncharged or lower-charged insulin formulations,an active flux enhancement through the micropores can be effected bycoupling a sonic field or sonophoresis, which may include frequenciesnormally described as ultrasonic, to help push the insulin into thetissues. An additional feature of the sonic field is its ability toenhance the permeability of the various barriers within the viabletissues letting the insulin reach a larger volume of tissue over whichthe desired absorption into the blood stream can take place. Modulatingthe sonic energy has been shown to be very effective in modulating thetotal flux of a compound through the micropores into or through thedeeper tissues, providing a second means of developing a bolus deliverysystem.

[0354] The exact pathways of absorption of insulin when given as asubcutaneous injection are still a subject of some debate. One of thereasons this is still unclear is the widely varying levels ofbio-availability demonstrated within a population, or even the samesubject, on an injection-by-injection basis. One hypothesized pathway isthe direct absorption through the capillaries and into the blood stream.A method for enhancing this process is to couple electroporation withthe surface poration, where the electroporation has been specificallyoptimized to work in the region of the capillary endothelial membranes,creating temporarily, a large number of openings to enhance this directabsorption. As with the iontophoresis and sonophoresis describedpreviously, the total voltage amplitude levels of the electroporationsystem required to effect this type of electroporation within thesetissue layers beneath the outer surface are often lower than needed topenetrate through an intact outer surface due to the reduction of thebulk impedance of the outer layer of the biological membrane.

EXAMPLE 47

[0355] Delivery of microparticles: The use of liposomes, lipidcomplexes, microspheres including nanospheres, PEGellated compounds(compounds combined with polyethylene glycol) and other microparticlesas part of a drug delivery system is well developed for many differentspecific applications. In particular, when dealing with a compound whichis easily broken down by the endogenous components in the body's tissuessuch as protease, nuclease, or carbohydrase enzymes in the skin,tissues, the macrophages or other cells present in the blood stream orlymph, increases in bio-availability and/or sustained release canfrequently be realized by utilizing one of these techniques. Currently,once one has applied one of these techniques, the formulation isgenerally delivered via some type of injection. The present invention,by creating micropores through a biological membrane (e.g., the skin ora mucous membrane) and into the body to a selected depth, allows thistype of microparticle to be delivered through the skin or mucosa. Asdescribed in the insulin example above, microporation, electroporation,iontophoresis, sonic energy, enhancers, as well as mechanicalstimulation of the site such as pressure or massage may be combined inany combination to enhance the delivery and/or uptake of a specificformulation. In the case of some engineered microparticles, the poresmay have an optimal depth designed to bypass certain biologically activezones or place the particle within the zone of choice. For somemicroparticle delivery systems, the energy incident upon the particlesafter they have been delivered into or through the tissues beneath thesurface may be used to trigger the accelerated release of the activecompound, thereby allowing the external control of the flux rate of thetherapeutic substance.

EXAMPLE 48

[0356] Microparticles for implantable analyte monitoring: Anotherapplication of microparticles is to deliver a particle not as atherapeutic agent but as a carrier of a probe compound which could beinterrogated non-invasively, for example, via electromagnetic radiationfrom an external reader system to obtain information regarding thelevels of a specific analyte in the body. One example is to incorporatein a porous microsphere a glucose specific fluorophore compound which,depending on the levels of glucose present in the surrounding tissues,would alter its fluorescent response in either amplitude, wavelength, orfluorescent lifetime. If the fluorophore was designed to be active withan excitation wavelength ranging from 700 nm to 1500 nm, a low costinfrared light source such as an LED or laser diode could be used tostimulate its fluorescent response, which would similarly be in thisrange of from 700 nm to 1500 nm. At these wavelengths, the skin andmucosal tissues absorb very little and would therefore allow a simplesystem to be built along these lines.

[0357] Glucose is one candidate analyte, for which experimental lifetimefluorescence probes have been developed and incorporated intosubcutaneously inserted polymer implants which have been successfullyinterrogated through the skin with optical stimulation and detectionmethods. It would merely require the reformulation of these experimentalimplants into suitably sized microparticles to allow the delivery intoor through the viable tissue layers via the micropores. However, anyanalyte could be targeted, and the method of interrogating the deliveredmicroparticles could be via magnetic or electric field rather thanoptical energy.

EXAMPLE 49

[0358] Delivery of a Vaccine

[0359] A bacterial, viral, toxoid or mixed vaccine is prepared as asolid, liquid, suspension, or gel as required. This formulation couldinclude any one or combination of peptides, proteins, carbohydrates,DNA, RNA, entire microorganisms, adjuvants, carriers and the like. Aselected site of an individual is porated (skin or mucous membrane)according to the procedures described above in Example 45 and thevaccine is applied to the porated site. The depth of the micropores maydepend on the type of vaccine delivered. This delivery can be aided withelectroporation, iontophoresis, magnetic or sonic energy, enhancers, aswell as mechanical stimulation of the site such as pressure or massageaccording to the procedures described above and/or use electroporation,iontophoresis, magnetic or sonic energy, enhancers, as well asmechanical stimulation of the site such as pressure or massage toenhance cellular uptake. Additional or reinforcing doses can bedelivered in the same manner to achieve immunization of the individual.

EXAMPLE 50

[0360] Delivery of Testosterone: A commercially available testosteronepatch, the Androderm^(R)patch from TheraTech, Inc., was used in a set ofexperiments to evaluate the benefits of microporation as it applies tothe delivery of this permeant. A hypergonadic male subject went offAndroderm therapy for two days, after which a series of venous bloodsamples were drawn during the subsequent 24 hour period to establishthis subject's baseline levels of testosterone. Two 2.5 mg Androdermpatches were then installed as recommended by the manufacturer and asimilar set of venous blood sample were drawn to measure thetestosterone levels when the only transdermal flux enhancement methodbeing used was the chemical permeation enhancers contained in the patch.After two more days of a washout period, two 2.5 Androderm patches werethen similarly installed, but prior to the installation, the skinsurface at the target sites was porated with 72 micropores per site,each pore measuring approximately 80 μm in width and 300 μm in lengthand extending to a depth of 80 to 120 μm. For the porated delivery phasea similar set of venous blood sample were drawn to measure thetestosterone. The data from all three of these twenty four hour periodsis shown in the FIG. 35 titled ‘Effects of Microporation on TransdermalTestosterone Delivery’. A noteworthy feature of these data is that whenthe microporations are present, the testosterone levels in the subjectsblood elevate much more rapidly, essentially preceding the rising edgeof the un-porated cycle by more than four hours. Looking at the slope ofand area under the curve we can calculate that more than a three-foldflux rate enhancement took place due to the microporations during thefirst four hours.

EXAMPLE 51

[0361] Delivery of Alprostadil: Alprostadil, or PGE1, is a prostaglandinused therapeutically to treat male erectile dysfunction via it'svasodilator behavior. The standard delivery mode for this drug is adirect injection into the base of the penis or via a suppositoryinserted into the urethra. A set of experiments were conducted with twohealthy male volunteers. Each subject had a site of 1 square cm on thebase of the penis shaft prepared by porating 12 to 36 micropores on thisarea, with the thermal poration parameters set to create pores roughly100 microns deep as measured from the surface of the skin. Aconcentrated solution of alprostadil was placed in a small reservoirpatch placed on the poration site, an ultrasonic transducer was thenplaced on the top of the reservoir and activated and the subject'serectile and other clinical responses were recorded on video tape. Bothsubjects developed a significant amount of engorgement of the penis,estimated as achieving 70% of more of a full erection at the doseapplied. In addition, a malar flush response to the systemic levels ofthe drug delivered was observed. Over a 30 to 60 minute delivery period,both subjects developed a profound malar flush extending from the face,neck, chest and arms. Both the erectile response and the malar flushprovide evidence of the delivery of a clinically active amount of thedrug, a well know vasodilator.

EXAMPLE 52

[0362] Delivery of Interferon: Interferons are proteins of approximately17-22,000 molecular weight, that are administered clinically to treat avariety of disease states, such as viral infections (e.g., hepatitis Band C), immune diseases (such as multiple sclerosis), and cancers (e.g.,hairy cell leukemia). Due to their protein nature, interferons mustcurrently be administered by injection, as they cannot be given orallyand are too large for traditional transdermal or transmucosal deliverymethods. To demonstrate delivery of an interferon via the microporationtechnique, a 100 microliter aliquot of alpha-interferon solutioncontaining interferon with a specific activity of 100 millioninternational units of interferon per mg dissolved in 1 ml of deliverysolution, is applied to a 1 square cm area of porated skin, porated to adepth of 150-180 μm, thus falling short of the capillary bed, on thethigh of a healthy human subject. Trials are run using either purelypassive diffusion and with the application of sonic energy to the regionat sufficient amplitude, frequency, and modulation thereof to acceleratethe migration of the interferon through pores into or through theunderlying tissues without causing deleterious heating of the interferonsolution. Venous blood draws are taken at various time intervals forboth trials, and are assayed for interferon levels usingradio-immunoassay and bioassay. Interferon is detected in the serum overthe 4 hour time period monitored. The interferon levels for thesonically enhanced delivery experiment are detected sooner than for thepassive experiment. In another experiment, the interferon isadministered in dry powder form directly to the micropores in theporated area of the skin. Interferon is detected in the serum using thesame techniques as described above. In another test, the interferonsolution is applied in a gel with or without a backing film to theporated tissue of the buccal mucosa. Venous blood is drawn and assayedfor interferon levels. Interferon is detected in the serum over the 3hour time period monitored. In another experiment, the interferon isincorporated into a tablet containing a bio-erodable matrix, with amucoadherent polymer matrix that provided contact of the tablet over thearea of buccal mucosa that is porated. Interferon is detected in theserum using the same techniques as described above.

EXAMPLE 53

[0363] Delivery of morphine: A solution of morphine is applied to aporated area on the volar forearm of the human subjects. A positivepressure gradient is used to provide a basal delivery rate of themorphine into the body, as determined by assay of venous blood draws atappropriate time intervals for the presence of morphine. A basal levelof morphine of approximately 3-6 ng/ml is achieved. Upon demand, anadditional pressure bolus is applied to result in a spike in thedelivery of the morphine. The additional pressure bolus is achieved inone test by use of ultrasound; or in another experiment by the use of apressure spike. This type of delivery, in which a basal level of themorphine is continuously applied, with spikes in morphine deliveryperiodically upon demand, is useful in treating chronic and breakthroughpain.

EXAMPLE 54

[0364] Delivery of a disease resistant DNA into a plant: The seeds of aselected corn plant are microporated. The seeds are placed in a solutionof a permeant formulation containing DNA that encodes disease resistanceproteins. Sonic energy is used, optionally, to enhance the delivery ofthe DNA into the corn seeds. The seeds are germinated and grown tomaturity. The resulting seeds of the mature corn plants now carry thedisease resistant gene.

EXAMPLE 55

[0365] Delivery of DNA into a plant: The seeds of a sugar beet aremicroporated. The seeds are placed in a solution of a permeantformulation containing DNA that encodes human growth hormone.Electroporation, iontophoresis, sonic energy, enhancers, as well asmechanical stimulation of the site such as pressure may be used toenhance the delivery of the DNA into the seeds. The seeds are germinatedand grown to maturity. The resulting mature beet plant can now beharvested and the human growth hormone extracted for subsequentpurification and clinical use.

EXAMPLE 56

[0366] An experiment was conducted wherein fluorescent dextranparticles, MW approximately 10,000 Daltons, were applied in an aqueoussolution by means of a reservoir patch over a one square cm of skin onthe volar forearm of a human subject where 36 micropores extendingapproximately 80 μm in depth were formed. The reservoir patch was leftin place for 5 minutes. The porated site and surrounding area wereimaged with a fluorescent video microscope to evaluate the penetrationof the permeant into the tissue. The fluorescence showed that within 5minutes significant permeation of dextran occurred more than 2 mm awayfrom the nearest micropore. The video assay system used 10 minutes latershowed further diffusion so that the fluorescent flush extended 10 mmfrom the pores. This experiment gives clear evidence that this techniqueallows delivery of permeants with molecular weights of 110,000.

1. A method for enhancing the transmembrane flux rate of a permeant into a selected site of an organism comprising the steps of enhancing the permeability of said selected site of the organism to said permeant by means of (a) porating a biological membrane at said selected site by means that form a micropore in said biological membrane, thereby reducing the barrier properties of said biological membrane to the flux of said permeant; and (b) contacting the porated selected site with a composition comprising an effective amount of said permeant, whereby the transmembrane flux rate of said permeant into the organism is enhanced.
 2. The method of claim 1 further comprising applying to said site of said organism an enhancer to increase the flux of said permeant into said organism.
 3. The method of claim 2 wherein said enhancer comprises sonic energy.
 4. The method of claim 3 wherein said sonic energy is applied to said site at a frequency in the range of about 10 Hz to 1000 MHz, wherein said sonic energy is modulated by means of a member selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.
 5. The method of claim 2 wherein said enhancer comprises an electromagnetic field.
 6. The method of claim 5 wherein the electromagnetic field comprises iontophoresis.
 7. The method of claim 5 wherein the electromagnetic field comprises a magnetic field.
 8. The method of claim 2 wherein said enhancer comprises a mechanical force.
 9. The method of claim 2 wherein said enhancer comprises chemical enhancers.
 10. The method of claim 2 wherein any of the methods of claims 3, 4, 5, 6, 7, 8, or 9 may be applied in any combination thereof to increase the transmembrane flux rate of said permeant into or through said micropore.
 11. The method of claim 2, wherein said enhancers at said site are applied so as to increase the flux rate of the permeant into tissues surrounding the micropore.
 12. The method of claim 11, wherein said enhancer comprises sonic energy.
 13. The method of claim 12 wherein said sonic energy is applied to said site at a frequency in the range of about 10 Hz to 1000 MHz, wherein said sonic energy is modulated by means of a member selected from the group consisting of frequency modulation, amplitude modulation, phase modulation, and combinations thereof.
 14. The method of claim 11, wherein said enhancer comprises electroporation.
 15. The method of claim 11, wherein said enhancer comprises iontophoresis.
 16. The method of claim 11, wherein said enhancer comprises chemical enhancers.
 17. The method of claim 11, wherein said enhancer comprises a mechanical force.
 18. The method of claim 11 wherein said enhancer comprises a magnetic field.
 19. The method of claim 11, wherein said enhancer comprises any combination thereof of the methods of claims 12, 13, 14, 15, 16, 17, and
 18. 20. The method of claim 10 further comprising the method of claim
 19. 21. The method of claim 1 wherein said porating of said biological membrane in said site is accomplished by means selected from the group consisting of (a) ablating the biological membrane by contacting said site of said biological membrane with a heat source such that a micropore is formed in said biological membrane at said site; (b) puncturing said biological membrane with a micro-lancet calibrated to form a micropore; (c) ablating the biological membrane by a beam of sonic energy onto said biological membrane; (d) hydraulically puncturing said biological membrane with a high pressure jet of fluid to form a micropore and (e) puncturing said biological membrane with short pulses of electricity to form a micropore
 22. The method of claim 21 wherein said porating is accomplished by contacting said site, up to about 1000 μm across, with a heat source to conductively transfer an effective amount of thermal energy to said site such that the temperature of some of the water and other vaporizable substances in said site is elevated above their vaporization point creating a micropore to a selected depth in the biological membrane at said site.
 23. The method of claim 21 wherein said porating is accomplished by contacting said site, up to about 1000 μm across, with a heat source to conductively transfer an effective amount of thermal energy to said site such that the temperature of some of the tissue at said site is elevated to the point where thermal decomposition occurs creating a micropore to a selected depth in the biological membrane at said site.
 24. The method of claim 21 comprising treating at least said site with an effective amount of a substance that exhibits sufficient absorption over the emission range of a pulsed light source and focusing the output of a series of pulses from said pulsed light source onto said substance such that said substance is heated sufficiently to conductively transfer an effective amount of thermal energy to said biological membrane to elevate the temperature to thereby create a micropore.
 25. The method of claim 24 wherein said pulsed light source emits at a wavelength that is not significantly absorbed by said biological membrane.
 26. The method of claim 25 wherein said pulsed light source is a laser diode emitting in the range of about 630 to 1550 nm.
 27. The method of claim 25 wherein said pulsed light source is a laser diode pumped optical parametric oscillator emitting in the range of about 700 and 3000 nm.
 28. The method of claim 25 wherein said pulsed light source is a member selected from the group consisting of arc lamps, incandescent lamps, and light emitting diodes.
 29. The method of claim 21 further comprising providing a sensing system for determining when the micropore in the biological membrane has reached the desired dimensions.
 30. The method of claim 29 wherein said sensing system comprises light collection means for receiving light reflected from said site and focusing said reflected light on a detector for receiving said light and sending a signal to a controller wherein said signal indicates a quality of said light, and a controller coupled to said detector and to said light source for receiving said signal and for shutting off said light source when a preselected signal is received.
 31. The method of claim 29 wherein said sensing system comprises an electrical impedance measuring system which can detect the changes in the impedance of the biological membrane at different depths into the organism as the micropore is formed.
 32. The method of claim 21 further comprising cooling said site and adjacent tissues such that said site and adjacent tissues are in a cooled condition.
 33. The method of claim 32 wherein said cooling means comprises a Peltier device.
 34. The method of claim 21 further comprising, prior to porating said site, illuminating at least said site with light such that said site is sterilized.
 35. The method of claim 21 comprising contacting said site with a solid element, wherein said solid element functions as a heat source to conductively transfer an effective amount of thermal energy to said biological membrane to elevate the temperature to thereby create a micropore.
 36. The method of claim 35 wherein said heat source is constructed to modulate the temperature of said site to greater than 100° C. within about 10 nanoseconds to 50 milliseconds and then returning the temperature of said site to approximately ambient temperature within about 1 millisecond to 50 milliseconds and wherein a cycle of raising the temperature and returning to ambient temperature is repeated one or more times effective for porating the biological membrane to the desired depth.
 37. The method of claim 36 wherein said returning to approximately ambient temperature of said site is carried out by withdrawing said heat source from contact with said site.
 38. The method of claim 36 wherein the modulation parameters are selected to reduce sensation to the animal subject.
 39. The method of claim 31 further comprising providing means for monitoring electrical impedance between said solid element of claim 35 and said organism through said site and adjacent tissues and means for advancing the position of said solid element such that as said poration occurs with a concomitant change in impedance, said advancing means advances the solid element such that the solid element is in contact with said site during heating of the solid element, until the selected impedance is obtained.
 40. The method of claim 38 further comprising means for withdrawing said solid element from contact with said site wherein said monitoring means is capable of detecting a change in impedance associated with contacting a selected layer underlying the surface of said site and sending a signal to said withdrawing means to withdrawn said solid element from contact with said site.
 41. The method of claim 35 wherein said solid element is heated by delivering an electrical current through an ohmic heating element.
 42. The method of claim 35 wherein said solid element is formed such that it contains an electrically conductive component and the temperature of said solid element is modulated by passing a modulated electrical current through said conductive element.
 43. The method of claim 35 wherein said solid element is positioned in a modulatable magnetic field wherein energizing the magnetic field produces electrical eddy currents sufficient to heat the solid element.
 44. The method of claim 21 wherein said poration is accomplished by puncturing said site with a micro-lancet calibrated to form a micropore.
 45. The method of claim 21 wherein said poration is accomplished by a beam of sonic energy directed onto said site.
 46. The method of claim 21 wherein said poration is accomplished by hydraulically puncturing said biological membrane with a high pressure jet of fluid to form a micropore.
 47. The method of claim 21 wherein said poration is accomplished by puncturing said biological membrane with short pulses of electricity to form a micropore.
 48. The method of claim 1 wherein said permeant comprises nucleic acid.
 49. The method of claim 48 wherein said nucleic acid comprises a DNA.
 50. The method of claim 48 wherein said nucleic acid comprises RNA.
 51. The method of claim 1, wherein the micropore in the biological membrane extends into a portion of the outer layer of the biological membrane ranging from 1 to 30 microns in depth.
 52. The method of claim 1, wherein the micropore in the biological membrane extends through the outer layer of the biological membrane ranging from 10 to 200 microns in depth.
 53. The method of claim 1, wherein the micropore in the biological membrane extends into the connective tissue layer of the biological membrane ranging from 100 to 5000 microns in depth
 54. The method of claim 1, wherein the micropore in the biological membrane extends through the connective tissue layer of the biological membrane ranging from 1000 to 10000 microns in depth.
 55. The method of claim 1, wherein the micropore penetrates the biological membrane to a depth determined to facilitate desired activity of the selected permeant.
 56. The method of claim 1, wherein the permeant comprises a polypeptide.
 57. The method of claim 56, wherein the polypeptide is a protein.
 58. The method of claim 56, wherein the polypeptide comprises a peptide.
 59. The method of claim 58, wherein the peptide comprises insulin.
 60. The method of claim 58, wherein the peptide comprises a releasing factor.
 61. The method of claim 1, wherein the permeant comprises a carbohydrate.
 62. The method of claim 61, wherein the carbohydrate comprises heparin.
 63. The method of claim 1, wherein permeant comprises an analgesic.
 64. The method of claim 63, wherein the analgesic comprises an opiate.
 65. The method of claim 1, wherein the permeant comprises a vaccine.
 66. The method of claim 1, wherein the permeant comprises a steroid.
 67. The method of claim 1, wherein the permeant is associated with a carrier.
 68. The method of claim 67, wherein the carrier comprises liposomes.
 69. The method of claim 67, wherein the carrier comprises lipid complexes.
 70. The method of claim 67, wherein the carrier comprises microparticles.
 71. The method of claim 67, wherein the carrier comprises polyethylene glycol compounds.
 72. The method of claim 65 combined with the method of claim
 66. 73. The method of claim 1 wherein the permeant comprises a substance which has the ability to change its detectable response to a stimulus when in the proximity of an analyte present in the organism. 